Herbicide safener combinations for acetyl co-enzyme a carboxylase herbicide resistant plants

ABSTRACT

The present invention provides for compositions and methods for producing crop plants by using that are resistant to herbicides, and treatment with herbicide safener compositions. In particular, the present invention provides for wheat plants, plant tissues and plant seeds that contain altered acetyl-CoA carboxylase (ACCase) genes and proteins treatment with one or more acetyl-CoA carboxylase herbicides preferably form the aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemical families and a cloquintocet acid safener.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional ApplicationNo. 62/292,442 filed Feb. 8, 2016 which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides for compositions and methods for reducingweeds and treating crop plants that are resistant to herbicides. Inparticular, the present invention provides for plants, plant tissues andplant seeds that contain modified acetyl-CoA carboxylase (ACC) genes andproteins that confer resistance to ACCase herbicides and combinationherbicide safener treatments that potentiate this response for improvedyield and crop production.

BACKGROUND OF THE INVENTION

Wheat is grown worldwide and is the most widely adapted cereal. Commonwheats are used in a variety of food products such as bread, cookies,cakes, crackers, and noodles. In general the hard wheat classes aremilled into flour used for breads and the soft wheat classes are milledinto flour used for pastries and crackers. Wheat starch is used in thefood and paper industries, as laundry starches, and in other products.

The primary threat to commercial wheat production is weed competition,resulting in decreased grain yields and inferior grain quality. Althoughcultivation can be used to eliminate weeds, soil from tilled fields ishighly vulnerable to wind and water erosion. Due to ease of applicationand effectiveness, herbicide treatment is the preferred method of weedcontrol. Herbicides also permit weed control in reduced tillage ordirect seeded cropping systems designed to leave high levels of residueon the soil surface to prevent erosion. The most significant weedcompetition in wheat comes from highly related grasses, such as wild oatand jointed goatgrass, and it is difficult to devise effective chemicalcontrol strategies for problematic weed species related to thecultivated crop since they tend to share herbicide sensitivities. Oneapproach to solving this problem involves the development of herbicideresistant varieties. In this system, herbicide-is applied “in-crop” tocontrol weeds without injuring the herbicide-tolerant crop plants.

The development of herbicide resistance in plants offers significantproduction and economic advantages; as such the use of herbicides forcontrolling weeds or plants in crops has become almost a universalpractice. However, application of such herbicides can also result indeath or reduced growth of the desired crop plant, making the time andmethod of herbicide application critical or in some cases unfeasible.

Of particular interest to farmers is the use of herbicides with greaterpotency, broad weed spectrum effectiveness and rapid soil degradation.Plants, plant tissues and seeds with resistance to these compounds wouldprovide an attractive solution by allowing the herbicides to be used tocontrol weed growth, without risk of damage to the crop. One such classof broad spectrum herbicides are those compounds that inhibit theactivity of the acetyl-CoA carboxylase (ACC) enzyme in a plant. Suchherbicides are included in the aryloxyphenoxypropionate (FOP) andcyclohexanedione (DIM) chemical families. For example, wheat issusceptible to many ACC inhibiting herbicides that target monocotspecies, making the use of these herbicides to control grassy weedsalmost impossible.

Due to the importance of wheat as a crop plant on the world stage, thereis a need for wheat hybrids that are resistant to the inhibitory effectsof ACC herbicides, as well as combination therapy herbicide treatmentsto potentiate the weed killing while maintaining health of plants.thereby allowing for greater crop yield when these herbicides are usedto control grassy weeds.

SUMMARY OF THE INVENTION

The present invention provides for compositions and methods for wheatproduction by use of wheat plants that are resistant to herbicides, anda potentiating herbicide safener combination. In particular, the presentinvention provides for wheat plants, varieties, lines, and hybrids, aswell as plant tissues and plant seeds that contain altered acetyl-CoAcarboxylase (ACC) genes and proteins that are resistant to inhibition byherbicides that normally inhibit the activity of the ACC protein. Theseplants in combination with a herbicide safener treatment can increasetime to production as the potentiating treatment can provide resistanceto and ACCase herbicide even with only a single copy of the altered ACCprotein is present in the plant. This increases time to production ofcommercial crops as it is not necessary to continue breeding to obtain acopy of the mutation on each chromosome nor in each of the three genomeswhich could take years of breeding.

Cultivated wheat is susceptible to many ACC inhibiting herbicides thattarget monocot or grassy weed species. However, as described herein awheat genotype was created that exhibits tolerance to ACC inhibitingherbicides. Genetic analysis has identified genetic differences within amutant wheat germplasm that results in an ACC herbicide resistancephenotype.

In one embodiment, the present invention provides for one or more wheatplants whose germplasm comprises a mutation that renders the planttolerant to ACC herbicides. Moreover, in further embodiments theinvention relates to the offspring (e.g., F1, F2, F3, etc.) of a crossof said plant wherein the germplasm of said offspring has the samemutation as the parent plant. Therefore, embodiments of the presentinvention provide for wheat varieties/hybrids whose germplasm contains amutation, such that the phenotype of the plants is ACC herbicideresistance. In some embodiments, said offspring (e.g., F1, F2, F3, etc.)are the result of a cross between elite wheat lines, at least one ofwhich contains a germplasm comprising a mutation that renders the planttolerant to ACC herbicides.

In one embodiment, the present invention provides a wheat plant whereinsaid wheat plant germplasm confers resistance to inhibition by one ormore acetyl-CoA carboxylase herbicides at levels of said one or moreherbicides that would normally inhibit the growth of a wheat plant. Insome embodiments, said one or more acetyl-CoA carboxylase herbicides arefrom Aryloxyphenoxypropionate (FOPs), cyclohexanedione (DIMs), andphenylpyrazolin (DENs) chemical families. In some embodiments, saidwheat plant germplasm that confers resistance to inhibition by one ormore acetyl-CoA carboxylase herbicides comprises one or more mutationsin the acetyl-CoA carboxylase gene as found in AF28-A, AF26-B and/orAF10-D, (ATCC Nos. PTA-123074, PTA-123076 and PTA-123075). Each of theselines contains a single copy of the mutation in the A, B, and D genomesrespectively. Importantly, according to the invention, the acetyl-CoAcarboxylase herbicides are combined with a cloquintocet acid safener andthe single copy varieties are shown to be just as effectively resistanceas the multiple copy lines, or multiple genome containing lines.Surprisingly, other known safener compounds did not potentiate theresponse.

In another embodiment, the present invention provides a method ofcontrolling weeds in the vicinity of a wheat plant or population ofplants, comprising providing an effective amount of one or moreacetyl-CoA carboxylase herbicides and a an effective amount of acloquintocet acid safener, applying said one or more acetyl-CoAcarboxylase herbicides and safener to a field comprising a wheat plantor population of wheat plants, and controlling weeds in the vicinity ofsaid wheat plant or population of wheat plants such that weed growth isadversely affected by the application of said one or more herbicides andgrowth of said wheat plant or population thereof is not adverselyaffected. In some embodiments, said one or more acetyl-CoA carboxylaseherbicides are from aryloxyphenoxypropionate (FOP) and cyclohexanedione(DIM) chemical families. In some embodiments, said wheat plant orpopulations of wheat plants comprise one or more copies of a Ala to Valchange at amino acid position 2004 (as referenced by standard blackgrass references gi|199600899|emb|AM408429.1| andgi|199600901|emb|AM408430.1Sequence ID NOS:13, 14 15 and 16, see alsoFIG. 9)and further as found in AF28-A, AF26-B and/or AF10-D, (ATCC Nos.PTA-123074, PTA-123076 and/or PTA-123075). According to the inventionthe mutation need not be present in homozygous state, or even in eachgenome.

In another embodiment, the present invention provides a wheat hybrid,line or variety, wherein said wheat hybrid, line or variety comprisesgermplasm comprising one or more mutations in the acetyl-CoA carboxylasegene such that resistance to one or more acetyl-CoA carboxylaseherbicides is conferred to said hybrid, line or variety. In someembodiments, said wheat hybrid, line or variety is created byintrogression of a wheat germplasm that comprises said one or moremutations for conferring resistance to one or more acetyl-CoAcarboxylase herbicides. In some embodiments, said wheat hybrid, line orvariety is created by incorporation of a heterologous gene comprisingone or more mutations for conferring resistance to one or moreacetyl-CoA carboxylase herbicides.

In yet another embodiment the invention includes an herbicide andsafener composition comprising one more ACCase herbicides fromaryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemicalfamilies and a cloquintocet acid safener. The herbicide and safener maybe combined into a single composition applied as a single doseconcurrently or may be each applied sequentially, in any order.According to the invention, in a herbicide safener composition caninclude from about 50 grams active Accase herbicide per kilogram (gai/kg) to about 600 g ai/kg, with respect to the total composition; andfrom about 50 g ai/kg to about 600 g ai/kg, with respect to the totalcomposition, of cloquintocet acid. Additional components such assurfactants, buffers and other functional components may be present.

In some embodiments, the wheat plants treated include nucleic acidsequences from wheat which encode acetyl-CoA carboxylase. According tothe invention, wild-type sequences encoding acetyl-CoA carboxylase havebeen identified from the B, D, and A genome, (SEQ ID NOS: 1, 2, and 3,respectively). Further, mutations each genome have been identified whichprovide resistance to acetyl-CoA carboxylase herbicide, SEQ ID NOS: 4,5, and 6, respectively. The mutation represents a change from Ala to Valat amino acid position 2004 (as referenced by standard black grassreferences gi|199600899|emb|AM408429.1| andgi|199600901|emb|AM408430.1Sequence ID NOS:13, 14 15 and 16, see alsoFIG. 9) for the each genome, A genome, (SEQ ID NO: 8); B genome, (SEQ IDNO: 10), D genome, (SEQ ID NO: 12). The invention also includes aminoacids encoded by these sequences, including SEQ ID NO: 7, 8, 9, 10, 11or 12, as well as conservatively modified variants, and fragments whichretain ACCase activity as well as the mutants which provide resistanceto acetyl-CoA carboxylase herbicide.

Thus wheat plants treated according to the invention include apolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence comprising SEQ ID NO:7, 9 or11 and SEQ ID NOS 8, 10, or 12 and (b) the amino acid sequencecomprising at least 90%, 95% or 99% sequence identity to SEQ ID NO:7, 9,11 or SEQ ID NOS: 8, 10, or 12 wherein said polypeptide has ACCaseactivity or provides resistance to acetyl-CoA carboxylase herbicide.

The invention also includes use of wheat and other cereal plants thatcomprise a nucleotide sequence that is at least 70% homologous, at least80% homologous, at least 85% homologous, at least 90% homologous, atleast 95% homologous, at least 97% homologous, or at least 99%homologous to the acetyl-CoA carboxylase sequence of SEQ ID NO:1, 2, 3,4, 5, or 6 or as found in AF28-A, AF26_B, and/or AF10-D (ATCC Nos.PTA-123074, PTA-123076 and/or PTA-123075). In some embodiments, theacetyl-CoA carboxylase sequence encodes or comprises one or more aminoacid substitutions, for example Ala2004Val as found in SEQ ID NOS: 8,10, or 12.

In yet another embodiment, the invention provides for the use ofgenetically modified wheat plants incorporating a heterologousnucleotide construct including SEQ ID NOS: 1, 2, 3, 4, 5, or 6 operablylinked to regulatory sequences such as expression cassettes, inhibitionconstructs, plants, plant cells, and seeds. The genetically modifiedplants, plant cells, and seeds of the invention may exhibit phenotypicchanges, such as modulated ACCase or mutant ACCase levels.

DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph of the first herbicide tolerant plant discovered.This plant survived two lethal applications of clethodim herbicide.

FIG. 2 is a photograph of M3 plants grown from seed of two M2 parents.Plants were treated with two sequential rates of a lethal dose ofquizalofop. The plants on the left survived both herbicide applications;the plants on the right died after one application.

FIG. 3 is a photograph of a dose response study exhibiting the increasedtolerance of selected mutant plants to quizalofop herbicide in the M3generation compared to non-mutagenized Hatcher winter wheat. Column 1,3, and 4 are plants selected for increased herbicide tolerance; column 2is non-mutagenized winter wheat.

FIG. 4 are the sequences of the ACCase genes from the A, B and D genomesand the mutant AF28 A ACCase gene, the mutant AF26-B and mutant AF10-Dgene.

FIG. 5 is a graph depicting visual injury of M2-derived M3 mutantsscreened with quizalofop. Values below the horizontal line are differentthan the non-mutagenized check, represented by the far left bar.

FIG. 6 is a graph depicting a dose response trial with quizalofopcomparing the non-mutagenized check, represented by the left bar, withM2-selected M3 accessions.

FIG. 7 is a graph showing a comparison of wild type and mutant ACCasesequences in wheat A, B, D genomes, including a newly discoverednon-synonymous SNP in each mutant sequence.

FIG. 8 is a graph showing a comparison of ACCase enzyme tolerance toincreasing quizalofop concentrations.

FIG. 9 shows alignment of the sequences of the invention to black grassreference sequence and to each other.

FIG. 10 is a graph of height data as a percent change from untreatedcontrol. Letters indicate differences within lines at p<0.05.

FIG. 11 is a graph showing yield data as a percent change from untreatedcontrol. Letters indicate differences within lines at p<0.05.

FIG. 12 is a graph showing the visual rating data on a scale of 0 (noinjury) to 10 (complete mortality). Letters indicate differences withinlines at p<0.05.

DEFINITIONS

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided. Units, prefixes, and symbols maybe denoted in their SI accepted form. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Numeric ranges are inclusive of the numbersdefining the range and include each integer within the defined range.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.Unless otherwise provided for, software, electrical, and electronicsterms as used herein are as defined in The New IEEE Standard Dictionaryof Electrical and Electronics Terms (5th edition, 1993). The termsdefined below are more fully defined by reference to the specificationas a whole.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or conservatively modified variants of the aminoacid sequences. Because of the degeneracy of the genetic code, a largenumber of functionally identical nucleic acids encode any given protein.For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinthat encodes a polypeptide also, by reference to the genetic code,describes every possible silent variation of the nucleic acid. One ofordinary skill will recognize that each codon in a nucleic acid (exceptAUG, which is ordinarily the only codon for methionine; and UGG, whichis ordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide of the present invention isimplicit in each described polypeptide sequence and is within the scopeof the present invention.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W.H. Freeman and Company. Referenceto any sequence herein shall be interpreted to include conservativelymodified variants.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as are present in some plant, animal, andfungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)).

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentsthat normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment; or (2)if the material is in its natural environment, the material has beensynthetically (non-naturally) altered by deliberate human interventionto a composition and/or placed at a location in the cell (e.g., genomeor subcellular organelle) not native to a material found in thatenvironment. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural state. Forexample, a naturally occurring nucleic acid becomes an isolated nucleicacid if it is altered, or if it is transcribed from DNA which has beenaltered, by means of human intervention performed within the cell fromwhich it originates. See, e.g., Compounds and Methods for Site DirectedMutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In VivoHomologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., apromoter) becomes isolated if it is introduced by non-naturallyoccurring means to a locus of the genome not native to that nucleicacid. Nucleic acids which are “isolated” as defined herein, are alsoreferred to as “heterologous” nucleic acids.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or cDNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

Unless otherwise stated, the term “ACCase nucleic acid” means a nucleicacid comprising a polynucleotide (an “ACCase polynucleotide”) encodingan ACCase polypeptide with ACCase activity and includes allconservatively modified variants, homologs paralogs and the like. An“ACCase gene” is a gene of the present invention and refers to aheterologous genomic form of a full-length ACCase polynucleotide.

As used herein, the term “plant” can include reference to whole plants,plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells,seeds and progeny of same. Plant cell, as used herein, further includes,without limitation, cells obtained from or found in: seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen, and microspores. Plant cellscan also be understood to include modified cells, such as protoplasts,obtained from the aforementioned tissues. The class of plants which canbe used in the methods of the invention is generally as broad as theclass of higher plants amenable to transformation techniques, includingboth monocotyledonous and dicotyledonous plants. Particularly preferredplants include maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, and millet.

As used herein, “polynucleotide” or includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons as “polynucleotides” as thatterm is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides are not entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination,and they may be circular, with or without branching, generally as aresult of posttranslation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods, aswell. Further, this invention contemplates the use of both themethionine-containing and the methionine-less amino terminal variants ofthe protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such as Agrobacterium or Rhizobium. Examples of promotersunder developmental control include promoters that preferentiallyinitiate transcription in certain tissues, such as leaves, roots, orseeds. Such promoters are referred to as “tissue preferred”. Promoterswhich initiate transcription only in certain tissue are referred to as“tissue specific”. A “cell type” specific promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” or “repressible”promoter is a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most environmentalconditions.

As used herein “recombinant” or “genetically modified” includesreference to a cell or vector, that has been altered by the introductionof a heterologous nucleic acid or that the cell is derived from a cellso modified. Thus, for example, recombinant or genetically modifiedcells express genes that are not found in identical form within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under-expressed or not expressed atall as a result of deliberate human intervention. The term “recombinant”or “genetically modified” as used herein does not encompass thealteration of the cell or vector by naturally occurring events (e.g.,spontaneous mutation, natural transformation/transduction/transposition)such as those occurring without deliberate human intervention.

As used herein, a “expression cassette” is a nucleic acid construct,generated recombinantly or synthetically, with a series of specifiednucleic acid elements which permit transcription of a particular nucleicacid in a host cell. The recombinant expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA,virus, or nucleic acid fragment. Typically, the recombinant expressioncassette portion of an expression vector includes, among othersequences, a nucleic acid to be transcribed, and a promoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 50° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. for 20 minutes.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with .gtoreq.90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, 4, 5, or 6° C. lowerthan the thermal melting point (T_(m)); moderately stringent conditionscan utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lowerthan the thermal melting point (T_(m)); low stringency conditions canutilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acids Probes, Part I, Chapter 2,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995). In general a high stringency wash is 2×15 min in 0.5×SSCcontaining 0.1% SDS at 65° C.

As used herein, “transgenic plant” or “genetically modified plant”includes reference to a plant which comprises within its genome aheterologous polynucleotide. Generally, the heterologous polynucleotideis stably integrated within the genome such that the polynucleotide ispassed on to successive generations. The heterologous polynucleotide maybe integrated into the genome alone or as part of an expressioncassette. “Transgenic” or “genetically modified” is used herein toinclude any cell, cell line, callus, tissue, plant part or plant, thegenotype of which has been altered by the presence of heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” or “genetically modified” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” includes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence, a gap penalty is typically introducedand is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson, et al., Methods in Molecular Biology 24:307-331(1994). The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters. Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center forBiotechnology-Information www.hcbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993))and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993))low-complexity filters can be employed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g. chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, the term “variety” and “cultivar” refers to plants thatare defined by the expression of the characteristics resulting from agiven genotype or combination of genotypes, distinguished from any otherplant grouping by the expression of at least one of the characteristicsand considered as a unit with regard to its suitability for beingpropagation unchanged.

As used herein, the term “hybrid” refers to the offspring or progeny ofgenetically dissimilar plant parents or stock produced as the result ofcontrolled cross-pollination as opposed to a non-hybrid seed produced asthe result of natural pollination.

As used herein, the term “progeny” refers to generations of a plant,wherein the ancestry of the generation can be traced back to said plant.As used herein, the term “progeny” of an herbicide resistant plantincludes both the progeny of that herbicide resistant plant, as well asany mutant, recombinant, or genetically engineered derivative of thatplant, whether of the same species or a different species, where theherbicide resistant characteristic(s) of the original herbicideresistant plant has been transferred to the progeny plant.

As used herein, the term “plant tissue” includes differentiated andundifferentiated tissues of plants including those present in roots,shoots, leaves, pollen, seeds and tumors, as well as cells in culture(e.g., single cells, protoplasts, embryos, cellus, etc.). Plant tissuemay be in planta, in organ culture, tissue culture, or cell culture.

As used herein, the term “plant part” as used herein refers to a plantstructure or a plant tissue, for example, pollen, an ovule, a tissue, apod, a seed, and a cell. In some embodiments of the present inventiontransgenic plants are crop plants. As used herein, the terms “crop” and“crop plant” are used in their broadest sense. The term includes, but isnot limited to, any species of plant edible by humans or used as a feedfor animal or fish or marine animal, or consumed by humans, or used byhumans, or viewed by humans, or any plant used in industry or commerceor education.

As used herein, the term “elite germplasm” in reference to a plantrefers to hereditary material of proven genetic superiority.

As used herein, the term “elite plant,” refers to any plant that hasresulted from breeding and selection for superior agronomic performance.

As used herein, the term “trait” refers to an observable and/measurablecharacteristic of an organism. For example, the present inventiondescribes plants that are resistant to FOP and DIM herbicides.

As used herein, the terms “marker” and “DNA marker” and “molecularmarker” in reference to a “selectable marker” refers to a physiologicalor morphological trait that may be determined as a marker for its ownselection or for selection of other traits closely linked to thatmarker. For example, such a marker could be a gene or trait thatassociates with herbicide tolerance including, but not limited to,simple sequence repeat (SSR), single nucleotide polymorphism (SNP),genetic insertions and/or deletions and the like.

As used herein, the term “introgress” and “introgressing” and“introgression” refers to conventional (i.e. classic) pollinationbreeding techniques to incorporate foreign genetic material into a lineof breeding stock. For example, the present invention provides for wheatcrop plants introgressed with a mutant ACC gene for herbicide toleranceby crossing two plant generations.

As used herein, the term “wild-type” when made in reference to a generefers to a functional gene common throughout a plant population. Afunctional wild-type gene is that which is most frequently observed in apopulation and is thus arbitrarily designated the “normal” or“wild-type” form of the gene.

As used herein, the term “mutant” or “functional mutant” when made inreference to a gene or to a gene product refers, respectively, to a geneor to a gene product which displays modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. Thus, the terms “modified” and“mutant” when used in reference to a nucleotide sequence refer to annucleic acid sequence that differs by one or more nucleotides fromanother, usually related nucleotide acid sequence and the term“functional mutant” when used in reference to a polypeptide encodes bysaid “modified” or “mutant” nucleic acid refers to the protein orpolypeptide that retains activity. In the present application, the ACCmutant protein, “or functional mutant” thereof is an ACC gene thatretains its native activity to create essential amino acids.Additionally, a “modified” nucleotide sequence is interpreted as thatfound in the degenerate genetic code as known by those skilled in theart. For example, the genetic code is degenerate as there are instancesin which different codons specify the same amino acid; a genetic code inwhich some amino acids may each be encoded by more than one codon. It iscontemplated that the present invention may comprise such degeneracy(e.g., wherein a wheat hybrid comprises an ACC gene that is at least 70%homologous, at least 80% homologous, at least 85% homologous, at least90% homologous, at least 95% homologous, at least 97% homologous, or atleast 99% homologous to SEQ ID NO: 1, 2, 3, 4, 5, or 6 or that found inAF28-A, AF26-B and/or AF10-D, (ATCC Nos. PTA-123074, PTA-123076 and/orPTA-123075) as found in, for example, the wheat germplasm.

DETAILED DESCRIPTION OF THE INVENTION

Acetyl-CoA carboxylase (ACC) is a biotinylated enzyme that catalyzes thecarboxylation of acetyl-CoA to produce malonyl-CoA. This carboxylationis a two-step, reversible reaction consisting of the ATP-dependentcarboxylation of the biotin group on the carboxyl carrier domain bybiotin-carboxylase activity followed by the transfer of the carboxylgroup from biotin to acetyl-CoA by carboxyl-transferase activity(Nikolau et al., 2003, Arch. Biochem. Biophys. 414:211-22). Acetyl-CoAcarboxylase is not only a key enzyme in plants for biosynthesis of fattyacids, a process that occurs in chloroplasts and mitochondria, but ACCalso plays a role in the formation of long-chain fatty acids andflavonoids, and in malonylation that occurs in the cytoplasm. There aretwo isoforms of ACC with the chloroplastic ACC accounting for more than80% of the total ACC activity (Herbert et al., 1996, Biochem. J.318:997-1006). Aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM)are two classes of chemicals that are known to selectively inhibitchloroplastic ACC in grasses (Rendina et al., 1990, J. Agric. Food Chem.38:1282-1287).

Seeds from a wheat variety were exposed to the chemical mutagen ethanemethylsulfonate and were planted and evaluated for tolerance to ACCherbicides. One of the genotypes, AF28-A, (SEQ ID NO:4) expressed highlevels of tolerance to each of the herbicides tested. It was furtherdemonstrated herein that crossing the AF28-A, AF26-B and/or AF10-D, withelite parent lines yielded good seed set and ACC herbicide resistance inprogeny plants.

As such, one embodiment of the present invention provides a plantgermplasm that contains altered ACC genes and proteins. The inventionincludes the use of an ACC herbicides in combination with a safener infields of hybrid plants to reduce the amount of monocot weed plantspresent in said crop field, wherein said hybrid germplasm comprises analtered ACC enzyme that confers resistance to ACC herbicides and saidweed plants are ACC herbicide susceptible. Preferred plants includewheat, rice and barley or other monocot cereal plants with an analogousmutation. According to the invention, the combination of herbicide andsafener provides for good resistance and weed killing even when themutation is in a single copy. Thus obviating the need for additionalbreeding to develop plants that are homozygous for the mutation or forplants which have the mutation on each of the A, B, and D genome of theplant.

Thus the invention includes obtaining and planting a wheat variety withone or more mutations in the ACCase gene that confer resistance to anACCase herbicide. In a preferred embodiment the mutation is an Ala toVal substitution at position 2004 of the ACCase protein. In anotherpreferred embodiment the wheat variety is one or more of AF28-A, AF26-B,and/or AF10D or a descendant thereof. The plants are then treated withan effective amount of an ACCase herbicide and a cloquintocet acid (CQCacid) safener. The herbicide can be combined in a single composition orused sequentially in either order.

ACCase Herbicide

In some embodiments, said one or more acetyl-CoA carboxylase herbicidesare from Aryloxyphenoxypropionate (FOPs), cyclohexanedione (DIMs),and/or phenylpyrazolin (DENs) chemical families. In some embodiments ofthe invention, the at least one herbicide used in the method is anacetyl coenzyme A carboxylase (ACCase) inhibitor. In some embodiments,the at least one herbicide is selected from the group of: alloxydim,butroxydim, cloproxydim, profoxydim, sethoxydim, clefoxydim, clethodim,cycloxydim, tepraloxydim, tralkoxydim, chloraizfop, clodinafop, clofop,cyhalofop, diclofop, fenoxaprop, fenthiaprop, fluazafop-butyl,fluazifop, haloxyfop, isoxapyrifop, metamifop, propaquizafop,quizalofop, trifop and pinoxaden.

Herbicides that act as acetyl-coenzyme A carboxylase (ACCase) inhibitorsinterrupt lipid biosynthesis in plants, which can lead to membranedestruction actively growing areas such as meristematic tissue. ACCaseinhibitors are exemplified by the aryloxyphenoxypropionate (APP)chemical family, also known as FOPS, and the cyclohexandione (CHD)family, also known as DIMs.

Accordingly, embodiments of the invention are directed to plantsselected for resistance to ACCase inhibitor herbicides and methods ofidentifying the same. In some embodiments, the plant is resistant to acyclohexanedione herbicide, an aryloxyphenoxy proprionate herbicide, aphenylpyrazoline herbicide, or mixtures thereof. In some embodiments,the plant is resistant to at least one herbicide selected from the listprovided in Table A.

TABLE A Acetyl Coenzyme A Carboxlyase Inhibitors Herbicide Class(Synonyms Active Name Synonyms Example Products CyclohexanedionesAlloxydim Carbodimedon, Fervin, Kusagard CHDs, DIMs) Zizalon, BAS 90210HButroxydim Butoxydim Falcone Clethodim Cletodime Select; Prism;Centurion; Envoy Cloproxydim Selectone Cycloxydim BAS 517H, BAS 517Focus, Laser; Stratos Profoxydim Clefoxydim Aura BAS 625 H SethoxydimCyethoxydim Poast; Rezult; Vantage; Checkmate, Expand, Fervinal,Grasidim, Sertin Tepraloxydim Caloxydim Aramo; Equinox TralkoxydimTralkoxydime; Achieve; Splendor; Tralkoxidym Grasp AryloxyphenoxyChlorazifop Propiomates (APPs, Clodinafop Discover, Topik FOPs) ClofopFenofibric Acid Alopex Cyhalofop Barnstorm; Clincher DiclofopDichlorfop; Illoxan Hoelon; Hoegrass; Illoxan Fenoxaprop Fenoxaprop-POption; Acclaim Fusion w/Fluazifop Fenthiaprop Fenthioprop; Taifun;Joker; Hoe Fentiaprop 35609 Fluzifop Fluazifop-P Fusilade DX; Fusionw/Fenoxaprop Haloxyfop Haloxyfop-P Edge; Motsa Verdict; GallantIsozapyrifop HOK-1566; RH 0898 Metamifop Propaquixafop Correct; Shogun;Agil Quizalofop Quizalofop-P; Quizafop Assure; Targa TrifopPhenylpyrazoline Pinoxaden Only known ACCase Axial (DENs) inhibitor inits class

Herbicidal cyclohexanediones include, but are not limited to, sethoxydim(2-[1-(ethoxyimino)-butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cylohexen-1-one,commercially available from BASF (Parsippany, N.J.) under thedesignation POAST®), clethodim((E,E)-(.+−.)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylth-io)propyl]-3-hydroxy-2-cyclohexen-1-one;available as SELECT™ from Chevron Chemical (Valent) (Fresno, Calif.)),cloproxydim((E,E)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one; available as SELECTONE™ fromChevron Chemical (Valent) (Fresno, Calif)), and tralkoxydim(2-[1-(ethoxyimino)propyl]-3-hydroxy-5-mesitylcyclohex-2-enone,available as GRASP™ from Dow Chemical USA (Midland, Mich.)). Additionalherbicidal cyclohexanediones include, but are not limited to,clefoxydim, cycloxydim, and tepraloxydim.

Herbicidal aryloxyphenoxy proprionates and/or aryloxyphenoxypropanoicacids exhibit general and selective herbicidal activity against plants.In these compounds, the aryloxy group can be phenoxy, pyridinyloxy orquinoxalinyl. Herbicidal aryloxyphenoxy proprionates include, but arenot limited to, haloxyfop((2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]-propanoicacid), which is available as VERDICT™ from Dow Chemical U.S.A. (Midland,Mich.)), diclofop (((.+−.)-2-[4-(2,4-dichlorophenoxy)-phenoxy]propanoicacid), available as HOELON™ from Hoechst-Roussel Agri-Vet Company(Somerville, N.J.)), fenoxaprop((.+−.)-2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxy]propanoic acid;available as WHIP™ from Hoechst-Roussel Agri-Vet Company (Somerville,N.J.)); fluazifop((.+−.)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoicacid; available as FUSILADE™ from ICI Americas (Wilmington, Del.)),fluazifop-P((R)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid;available as FUSILADE 2000.™ from ICI Americas (Wilmington, Del.)),quizalofop ((.+−.)-2-[4-[(6-chloro-2-quinoxalinyl)-oxy]phenoxy]propanoicacid; available as ASSURE® from E.I. DuPont de Nemours (Wilmington,Del.)), and clodinafop.

Analogs of Herbicidal Cyclohexanediones or Herbicidal AryloxyphenoxyProprionates or Herbicidal Phenylpyrazolines.

Included among the ACCase inhibitors are herbicides that arestructurally related to the herbicidal cyclohexanediones, herbicidalaryloxyphenoxy proprionates, or herbicidal phenylpyrazolines, as hereindisclosed, such as, for example, analogs, metabolites, intermediates,precursors, salts, and the like.

CQC Acid Safener and Herbicide Safener Compositions

The methods and herbicide compositions described herein include the useof cloquintocet acid (CQC acid) or a salt thereof CQC acid is aherbicide safener and has the following chemical structure:

CQC acid functions as a herbicide safener by reducing the phytotoxiceffects of the herbicide on crops to which it is applied. In someembodiments the herbicide safener used in the herbicide compositionsdescribed herein may comprise a salt of cloquintocet acid containing oneor more cations selected from sodium, potassium, and the class of organoammonium cations wherein the organo ammonium cations may have from 1 toabout 12 carbon atoms. Exemplary organo ammonium cations include, forexample, isopropyl ammonium, diglycol ammonium(2-(2-aminoethoxyl)ethanol ammonium), dimethyl ammonium, diethylammonium, triethyl ammonium, monoethanol ammonium, dimethylethanolammonium, diethanol ammonium, triethanol ammonium, triisopropanolammonium, tetramethyl ammonium, tetraethylammonium,N,N,N-trimethylethanol ammonium (choline), andN,N-bis-(3-aminopropyl)methyl ammonium (BAPMA).

When combined with the herbicide in a composition, the herbicide safenermay contain, with respect to the total composition, from about 50 gae/kg to about 600 g ae/kg of cloquintocet acid or a salt thereof. Insome embodiments the herbicide safener may comprise from about 50 gae/kg to about 300 g ae/kg, from about 50 g ae/kg to about 200 g ae/kg,from about 50 g ae/kg to about 150 g ae/kg, from about 50 g ae/kg toabout 125 g ae/kg, from about 50 g ae/kg to about 100 g ae/kg, fromabout 50 g ae/kg to about 80 g ae/kg, or from about 50 g ae/kg to about70 g ae/kg of cloquintocet acid or a salt thereof. In some embodimentsthe herbicide safener may contain from about 100 g ae/kg to about 600 gae/kg, from about 150 g ae/kg to about 600 g ae/kg, from about 200 gae/kg to about 600 g ae/kg, from about 200 g ae/kg to about 500 g ae/kg,from about 200 g ae/kg to about 400 g ae/kg, from about 200 g ae/kg toabout 350 g ae/kg, from about 200 g ae/kg to about 300 g ae/kg, fromabout 200 g ae/kg to about 250 g ae/kg, or from about 200 g ae/kg toabout 225 g ae/kg of cloquintocet acid or a salt thereof. In someembodiments the herbicide safener may contain from about 300 g ae/kg toabout 600 g ae/kg, from about 350 g ae/kg to about 500 g ae/kg, fromabout 400 g ae/kg to about 500 g ae/kg, or from about 425 g ae/kg toabout 475 g ae/kg of cloquintocet acid or a salt thereof.

In some embodiments the weight ratio, on an ae basis, of thecloquintocet acid safener or salt thereof, to the one or more herbicideactive ingredients in the herbicide compositions described herein mayrange from about 10:1 to about 1:10, from about 5:1 to about 1:5, fromabout 4:1 to about 1:4, from about 3:1 to about 1:3, or from about 2:1to about 1:2.

In some embodiments the herbicide compositions described herein furtherincludes a dispersant such as a lignosulfonate salt. Examples oflignosulfonate salts include sodium lignosulfonates and/or calciumlignosulfonates. Examples of lignosulfonate salt dispersants suitablefor use with the herbicide compositions described herein includePolyfon®. H, O, T, and F, Kraftsperse® 25M and Reax® 88B, 825 which areall available from MeadWestvaco (Richmond, Va.), and Borresperse® NA, CAand 3A which are available from Borregaard LignoTech (Houston, Tex.).

The dispersants used in the herbicide compositions described herein maycomprise from about 30 g/kg to about 250 g/kg, with respect to the totalcomposition. In some embodiments the herbicide compositions describedherein may include from about 30 g/kg to about 230 g/kg, from about 30g/kg to about 210 g/kg, from about 30 g/kg to about 190 g/kg, from about30 g/kg to about 170 g/kg, from about 30 g/kg to about 150 g/kg, fromabout 30 g/kg to about 130 g/kg, from about 30 g/kg to about 110 g/kg,from about 30 g/kg to about 90 g/kg, from about 30 g/kg to about 70g/kg, from about 30 g/kg to about 50 g/kg, or from about 30 g/kg toabout 40 g/kg of the dispersants. In some embodiments the herbicidecompositions described herein may also include from about 50 g/kg toabout 250 g/kg, from about 75 g/kg to about 250 g/kg, from about 75 g/kgto about 225 g/kg, from about 75 g/kg to about 200 g/kg, from about 75g/kg to about 175 g/kg, from about 100 g/kg to about 175 g/kg, fromabout 125 g/kg to about 175 g/kg, from about 145 g/kg to about 165 g/kg,from about 75 g/kg to about 125 g/kg, or from about 85 g/kg to about 115g/kg of the dispersants.

The compositions can further include anionic surfactants used in theherbicide compositions described herein may include at least one anionicsurfactant selected from those described, inter alia, in “McCutcheon'sDetergents and Emulsifiers Annual”, MC Publishing Corp., Ridgewood,N.J., 1998 and in the “Encyclopedia of Surfactants”, Vol. I-III,Chemical publishing Co., New York, 1980-81. Suitable anionicsurface-active agents may be selected from: salts of alkyl sulfates,such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, suchas calcium dodecylbenzenesulfonate; soaps, such as sodium stearate;alkylnaphthalene-sulfonate salts, such as sodiumdibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinate salts,such as sodium di(2-ethylhexyl) sulfosuccinate; salts of N-alkyl-N-fattyacid taurates; salts of mono- and dialkyl phosphate esters; and salts ofpolycarboxylates, such as sodium polycarboxylate.

In some embodiments, the anionic surfactant used in the herbicidecompositions described herein may include an N-alkyl-N-fatty acidtaurate surfactant such as, for example, sodium N-methyl-N-oleyl tauratewhich is available from Solvay Rhodia (Houston, Tex.) as Geropon® T-77.

In some embodiments, the anionic surfactant used in the herbicidecompositions described herein may include a sodium polycarboxylatesurfactant such as Geropon® T-36 (Solvay Rhodia).

The herbicide compositions described herein may include from about 10g/kg to about 100 g/kg, of at least one anionic surfactant. In someembodiments the herbicide compositions described herein may include fromabout 10 g/kg to about 90 g/kg, from about 10 g/kg to about 80 g/kg,from about 10 g/kg to about 70 g/kg, from about 10 g/kg to about 60g/kg, from about 10 g/kg to about 50 g/kg, from about 10 g/kg to about40 g/kg, from about 10 g/kg to about 30 g/kg, from about 20 g/kg toabout 50 g/kg, or from about 20 g/kg to about 40 g/kg, of at least oneanionic surfactant. In some embodiments the herbicide compositionsdescribed herein may include from about 20 g/kg to about 100 g/kg, fromabout 30 g/kg to about 100 g/kg, from about 40 g/kg to about 100 g/kg,from about 50 g/kg to about 100 g/kg, from about 60 g/kg to about 100g/kg, from about 70 g/kg to about 100 g/kg, or from about 70 g/kg toabout 90 g/kg, of at least one anionic surfactant.

Buffers useful in the herbicide compositions described herein generallyare very soluble in water (>20 weight %) and may include an organic orinorganic acid, or a salt thereof. Examples of buffers include ammoniumsulfate, diammonium phosphate, citric acid, potassium acetate, sodiumacetate, and combinations of the buffer with a clay.

The herbicide compositions described herein may include from about 50g/kg to about 250 g/kg o the buffer. In some embodiments the herbicidecompositions described herein may include from about 60 g/kg to about240 g/kg, from about 70 g/kg to about 230 g/kg, from about 80 g/kg toabout 220 g/kg, from about 90 g/kg to about 210 g/kg, from about 100g/kg to about 200 g/kg, from about 110 g/kg to about 190 g/kg, fromabout 120 g/kg to about 180 g/kg, from about 130 g/kg to about 170 g/kg,or from about 140 g/kg to about 160 g/kg of the buffer. In someembodiments the herbicide compositions described herein may also includefrom about 50 g/kg to about 250 g/kg, from about 50 g/kg to about 200g/kg, from about 50 g/kg to about 175 g/kg, from about 50 g/kg to about150 g/kg, from about 50 g/kg to about 125 g/kg, from about 50 g/kg toabout 100 g/kg, from about 50 g/kg to about 90 g/kg, from about 50 g/kgto about 80 g/kg, or from about 50 g/kg to about 70 g/kg of the buffer.

The composition may also contain inert ingredients that can serve as afiller, diluent, carrier, disintegrant, binding agent, processing aid,and/or flow aid and may help maintain the granules in a stable, state.These inert ingredients may include, for example, clays, starches,silicas, talc (hydrated magnesium silicate), palygorskites,pyrophyllites, attapulgus clay, kaolinite clay, bentonite clay,montmorillonite clay, illite clay and Fuller's earth, and diatomaceousearths such as diatomite, tripolite and kieselgur/kieselguhr,carbohydrates such as dextrines, alkylated celluloses, xanthum gums andguaseed gums, and synthetic polymers such as polyvinyl alcohols, sodiumpolyacrylates, polyethylene oxides, polyvinylpyrrolidones andurea/formaldehyde polymers. In the absence of effective inertingredients, dry granules may be physically unstable and slowlybreakdown forming a dust or powder. Many inert ingredients used inagricultural granule formulations generally have good water solubilityor dispersibility.

In some embodiments, the herbicide compositions described herein mayinclude a filler selected from one or more of a clay such as attapulgusclay, kaolinite clay, bentonite clay, montmorillonite clay, illite clayand Fuller's earth.

In some embodiments, the herbicide compositions described herein mayinclude a synthetic polymer selected from a polyvinyl alcohol, a sodiumpolyacrylate, a polyethylene oxide, a polyvinylpyrrolidone and aurea/formaldehyde copolymer like PergoPak® M which is available fromAlbemarle Corporation (Baton Rouge, La.), and mixtures thereof.

In some embodiments, the herbicide compositions described herein mayinclude a synthetic polymer such as a urea/formaldehyde copolymer likePergoPak® M, which may serve as a disintegrant and a processing aid.

Another aspect of the described herbicidal compositions includes addingone or more additional pesticide active ingredients, plant growthregulators, or safeners to the herbicidal compositions. These pesticideactive ingredients, plant growth regulators and safeners may include oneor more of an herbicide, an insecticide, a fungicide, a plant growthregulator or an herbicide safener.

Suitable additional herbicide safeners that may be added to theherbicidal composition described herein include benoxacor, benthiocarb,cloquintocet-mexyl, daimuron, dichlormid, dicyclonon, dimepiperate,fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole,Harpin proteins, isoxadifen-ethyl, mefenpyr-diethyl, mephenate, MG 191,MON 4660, naphthalic anhydride (NA), oxabetrinil, R29148 andN-phenyl-sulfonylbenzoic acid amides.

Suitable plant growth regulators that may be added to the herbicidalcomposition described herein include 2,4-D, 2,4-DB, IAA, IBA,naphthaleneacetamide, α-naphthaleneacetic acid, kinetin, zeatin,ethephon, aviglycine, 1-methylcyclopropene (1-MCP), ethephon,gibberellins, gibberellic acid, abscisic acid, ancymidol, flurprimidol,mefluidide, paclobutrazol, tetcyclacis, uniconazole, brassinolide,brassinolide-ethyl and ethylene.

In addition to the compositions and uses set forth above, the herbicidalcompositions described herein may be used in combination with one ormore additional compatible ingredients. Other additional compatibleingredients may include, for example, one or more agrochemical activeingredients, surfactants, dyes, fertilizers and micronutrients,pheromones and many other additional ingredients providing functionalutility, such as, for example, stabilizers, fragrants and dispersants.When the compositions described herein are used in combination withadditional active ingredients the presently claimed compositions can beformulated with the other active ingredient or active ingredients asherbicidal compositions, tank mixed in water with the other activeingredient or active ingredients for spray application or appliedsequentially with the other active ingredient or active ingredients inseparate or spray applications.

Surfactants conventionally used in the art of formulation and which mayoptionally be used in the present formulations are described, interalia, in “McCutcheon's Detergents and Emulsifiers Annual”, MC PublishingCorp., Ridgewood, N.J., 1998 and in “Encyclopedia of Surfactants”, Vol.I-III, Chemical publishing Co., New York, 1980-81. These surface-activeagents can be anionic, cationic or nonionic in character and can beemployed as emulsifying agents, wetting agents, suspending agents, orfor other purposes. Typical surface-active agents include salts of alkylsulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonatesalts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkyleneoxide addition products, such as nonylphenol-C18 ethoxylate; soaps, suchas sodium stearate; alkylnaphthalene-sulfonate salts, such as sodiumdibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinate salts,such as sodium di(2-ethylhexyl) sulfosuccinate; quaternary amines, suchas lauryl trimethylammonium chloride; block copolymers of ethylene oxideand propylene oxide; salts of mono and dialkyl phosphate esters.Oftentimes, some of these surfactants can be used interchangeably as anagricultural adjuvant, as a liquid carrier or as a surface active agent.

The herbicide compositions described herein offer acceptable herbicidalefficacy and crop safety when used to control weeds in cereal crops byspray application. The herbicide compositions may be added to or dilutedin an aqueous spray mixture for agricultural application such as forselective weed control in crop fields. Such compositions are typicallydiluted with an inert carrier, such as water, before application. Thediluted compositions, which are usually applied, for example, to weeds,the locus of weeds or the locus of where weeds may eventually emerge, insome embodiments contain from about 0.0001 to about 1 weight percent ofan active ingredient or from 0.001 to about 0.05 weight percent of anactive ingredient. The present compositions can be applied, for example,to weeds or their locus by the use of conventional ground or aerialsprayers, by addition to irrigation water and by other conventionalmeans known to those skilled in the art.

Methods of Controlling Weedy Grasses and Selectively GrowingHerbicide-Resistant Plants

Exclusion of undesirable weedy grasses can be accomplished by treatingthe area in which exclusive growth of resistant plant species isdesired, with herbicides to which resistance has been established incombination with the CQC acid safener. Accordingly, embodiments of theinvention also relate to methods of controlling weeds in the vicinity ofan herbicide-resistant plant identified by the methods disclosed herein,including: contacting at least one herbicide and safener to the weedsand to the herbicide-resistant plant, wherein the at least one herbicideand at least one safener of a CQC acid is contacted to the weeds and tothe plant at a rate sufficient to inhibit growth or cause death of anon-selected plant of the same species and/or of a weed species desiredto be suppressed. The non-selected plant typically is non-resistant tothe herbicide.

In some embodiments, the herbicide and safener can be contacted directlyto the herbicide-resistant plant and to the weeds. For example, theherbicide and safener can be dusted directly over theherbicide-resistant plant and the weeds. Alternatively, the herbicidecan be sprayed directly on the herbicide-resistant plant and the weeds.Other means by which the herbicide can be applied to theherbicide-resistant plant and weeds include, but are not limited to,dusting or spraying over an area or plot of land containing theherbicide-resistant plant and the weeds.

In some embodiments, the herbicide and/or safener can be contacted oradded to a growth medium in which the herbicide-resistant plant and theweeds are located. The growth medium can be, but is not limited to,soil, peat, dirt, mud, or sand. In other embodiments, the herbicideand/or safener can be included in water with which the plants areirrigated.

Typically, amounts of herbicide sufficient to cause growth or death of anon-resistant or non-selected plant ranges from about 2 μM or less toabout 100 μM or more of herbicide concentration. In some embodiments, asufficient amount of herbicide ranges from about 5 μM to about 50 μM ofherbicide concentration, from about 8 μM to about 30 μM of herbicideconcentration, or from about 10 μM to about 25 μM of herbicideconcentration. Alternatively, amounts of herbicide sufficient to causegrowth or death of a non-resistant plant ranges from about 25 grams ofactive ingredient per hectare (g ai ha.) to about 6500 g ai ha⁻¹ ofherbicide application. In some embodiments, a sufficient amount ofherbicide ranges from about 50 g ai ha⁻¹ to about 5000 g ai ha⁻¹ ofherbicide application, about 75 g ai ha⁻¹ to about 2500 g ai ha⁻¹ ofherbicide application, about 100 g ai ha⁻¹ to about 1500 g ai ha⁻¹ ofherbicide application, or about 250 g ai ha⁻¹ to about 1000 g ai ha⁻¹ ofherbicide application.

Safener can be combined with the herbicide or added as a separatetreatment. In some embodiments, the CQC acid or an agriculturallyacceptable salt or ester thereof, a) can be applied in an amount of from0.1 gram acid equivalent per hectare (g ae/ha) to 300 g ae/ha (e.g.,from 30 g ae/ha to 40 g ae/ha) and/or (b) can be applied in an amount offrom 1 grams active ingredient per hectare (g ai/ha) to 300 g ai/ha(e.g., from 30 g ai/ha to 40 g ai/ha). In some cases, (a) and (b) can beapplied in a weight ratio of from 65:1 to 1:5 (e.g., from 5:1 to 1:5, orfrom 2:1 to 1:2).

In embodiments of the invention, a method for controlling weeds in thevicinity of a herbicide-resistant plant is provided, wherein theherbicide-resistant plant is identified by the methods described inhereinafter, the method including: contacting at least one herbicide tothe weeds and to the herbicide-resistant plant, wherein the at least oneherbicide is contacted to the weeds and to the plant at a ratesufficient to inhibit growth of a non-selected plant of the same speciesor sufficient to inhibit growth of the weeds and contacting at least oneCQC acid safener to the weeds and to the herbicide-resistant planteither sequentially or concurrently. In some embodiments, theherbicide-resistant plant is resistant to an acetyl coenzyme Acarboxylase (ACCase) inhibitor. In some embodiments, the method includescontacting the herbicide and/or safener directly to theherbicide-resistant plant. In some embodiments, the method includescontacting the herbicide and or safener to a growth medium in which theherbicide-resistant plant is located.

Accase Resistant Plants and Varieties

In one embodiment, the present invention provides a plant withresistance to inhibition by ACC herbicides, singly or in conjunctionwith other resistance traits, for example insect resistance against thespotted stem borer Chilo partellus (Girijashankar et al., 2005, PlantCell Rep. 24:513-522, incorporated herein in its entirety). In someembodiments, for example, a wheat hybrid whose germplasm comprises asynthetic cryl Ac gene from Bacillus thuringiensis (Bt) is introgressedinto a wheat line whose germplasm confers resistance to ACC herbicides.As well, the incorporation of ACC herbicide resistance and insectresistance is accomplished via plant transgenesis into the same wheathybrid. One skilled in the art will recognize the various techniques asdescribed herein that are applicable to the incorporation of two or moreresistance attributes into the same plant.

In one embodiment, the present invention provides ACC herbicideresistance in plants comprising, for example, an ACC germplasmdesignated AF28-A, AF26-B and/or AF10-D, ATCC Nos. PTA-123074,PTA-123076 and/or PTA-123075 incorporated into elite varieties throughplant breeding and selection, thereby providing for the development ofherbicide tolerant plants that will tolerate the use of ACC inhibitingherbicides for weed control. Deployment of this herbicide tolerancetrait in the aforementioned plants allows use of these herbicides tocontrol monocot weeds that grow in the presence of these crops. In someembodiments, the incorporation of the ACC resistance trait into elitelines is via introgression, or classical breeding methods. In someembodiments, the incorporation of the ACC resistance gene into elitelines is via heterologous gene transgenesis with expression orinhibition constructs. In some embodiments, the invention provides aplant preferably wheat, wherein at least one ancestor of the wheat plantcomprises an ACC resistant gene from germplasm designated AF28-A,deposited under ATCC accession No: PTA-123074, PTA-123076 and/orPTA-123075 or a descendant thereof. In some embodiments, the ACCresistant herbicide gene includes a nucleic acid sequence that is atleast 70% homologous, at least 80% homologous, at least 85% homologous,at least 90% homologous, at least 95% homologous, at least 97%homologous, or at least 99% homologous to SEQ ID NO:4, or the ACCresistant herbicide gene as found in the AF28-A, deposited under ATCCaccession No: PTA-123074, PTA-123076 and/or PTA-123075 or a descendantthereof. In some embodiments, the ACC resistant herbicide gene is atleast 70% homologous, at least 80% homologous, at least 85% homologous,at least 90% homologous, at least 95% homologous, at least 97%homologous, or at least 99% homologous SEQ ID NO:4 or the ACC resistantherbicide gene as found in the AF28-A, deposited under ATCC accessionNo: PTA-123074, PTA-123076 and/or PTA-123075 or a descendant thereofcomprising an amino acid substitution Ala2004Val.

In some embodiments, ACC herbicide resistant germplasm is introgressedinto an elite plant line using classic breeding techniques. Examples ofclassical breeding methods for wheat, barley, rice and other monocotcereal plants can be found in, for example, Sleper and Poehlman, 2006,Breeding Field Crops, Fifth Edition, Blackwell Publishing, incorporatedherein in its entirety.

In one embodiment, the ACC herbicide resistant germplasm is introgressedinto a plant, preferably wheat that provides food for human consumption.In some embodiments, the ACC herbicide resistant germplasm isintrogressed into wheat plants that provide food for livestock (e.g.,poultry, cattle, swine, sheep, etc). In some embodiments, the ACCherbicide resistant germplasm is introgressed into wheat plants that areused in industrial processes such as ethanol production. In oneembodiment, the ACC herbicide resistant gene is introduced into theplant genome via transgenesis using vectors and technologies known inthe art.

In some embodiments, the present invention provides an ACC resistantgermplasm of a wheat plant part of line AF28-A, deposited under ATCCaccession No: PTA-123074, PTA-123076 and/or PTA-123075 or a descendantthereof and said wheat plant part is one or more of a pollen, an ovule,a tissue, a pod, a seed, and a cell. In one embodiment, the presentinvention provides an F1 hybrid whose germplasm comprises an ACCresistance gene as described herein. In some embodiments, the F1 hybridis a cross between two elite wheat lines, at least one of which containsa germplasm comprising an ACC resistance gene as described herein.

In one embodiment, the present invention provides methods forcontrolling weeds in a population of plants. In some embodiments,controlling the weeds comprises applying an ACC herbicide to saidpopulation of plants, such that weed growth is inhibited but plantgrowth is not adversely affected. In some embodiments, the ACC herbicidebeing applied is from the aryloxyphenoxypropionate (FOP) herbicidefamily including, but not limited to, clodinafop-propargyl,cyhalofop-butyl, diclofop-methyl, fenoxaprop-p-ethyl, fluazifop-b-butyl,haloxyfop-ethoxyethyl, haloxyfop-etotyl, haloxyfop-R-methyl,propaquizafop, quizalofop-p-ethyl and quizalo-P-refuryl compounds. Insome embodiments, the ACC herbicide being applied is from thecyclohexanediones (DIM) herbicide family including, but not limited to,alloxydim, butroxydim, clefoxydim, clethodim, cycloxydim, profoxydim,sethoxydim, tepraloxydim and tralkoxydim compounds. In some embodiments,the ACC herbicide being applied comprises a combination of compoundsfrom both FOP and DIM ACC herbicide families as disclosed herein.However, the present application is not limited to the ACC herbicideused, and a skilled artisan will appreciate that new ACC herbicides arebeing discovered at any given time that inhibit the ACC enzyme.

In one embodiment, the present invention provides for a plant (e.g., F1,F2, F3, F4, etc.) whose germplasm confers resistance to ACC herbicidesand resistance to one or more additional herbicides from one or moredifferent herbicide groups. For example, additional herbicide groupsused to inhibit weed growth, include, but are not limited to, inhibitorsof lipid synthesis (e.g., aryloxyphenoxypropionates, cyclohexanodeiones,benzofuranes, chloro-carbonic acids, phosphorodithioates,thiocarbamates), inhibitors of photosynthesis at photosystem II (e.g.,phenyl-carbamates, pyridazinones, triazines, triazinones, triazolinones,uracils, amides, ureas, benzothiadiazinones, nitriles,phenyl-pyridines), inhibitors of photosynthesis at photosystem I (e.g.,bipyridyliums), inhibitors of protoporphyrinogen oxidase (e.g.,diphenylethers, N-phenylphthalimides, oxadiazoles, oxyzolidinediones,phenylpyrazoles, pyrimidindiones, thiadiazoles), inhibitors ofcarotenoid biosynthesis (e.g., pyridazinones, pyridinecarboxamides,isoxazolidinones, triazoles), inhibitors of4-hydroxyphenyl-pyruvate-dioxygenase (e.g., callistemones, isoxazoles,pyrazoles, triketones), inhibitors of EPSP synthase (e.g., glycines),inhibitors of glutamine synthetase (e.g., phosphinic acids), inhibitorsof dihydropteroate synthase (e.g., carbamates), inhibitors ofmicrotubule assembly (e.g., benzamides, benzoic acids, dinitroanilines,phosphoroamidates, pyridines), inhibitors of cell division (e.g.,acetamides, chloroacetamides, oxyacetamides), inhibitors of cell wallsynthesis (e.g., nitriles, triazolocarboxamides) and inhibitors of auxintransport (e.g., phthalamates, semicarbazones). In some embodiments, thepresent invention provides F1 hybrids from elite plant lines thatcomprise resistance to one or more ACC herbicides alone, or inconjunction with, herbicide resistance to one or more of theaforementioned herbicide groups.

In one embodiment, the present invention provides use of a heterologousnucleotide sequence comprising SEQ ID NOS: 1, 2, 3, 4, 5, or 6 encodinga wild-type or mutant ACCase protein (SEQ ID NOS 7, 8, 9, 10, 11 or 12)for providing the selected agronomic trait of ACCase herbicideresistance. In one embodiment, the nucleotide sequence comprises amutant ACCase gene as found in the germplasm designated AF28-A, AF26-Band/or AF10-D, deposited under ATCC accession Nos. PTA-123074,PTA-123076 and/or PTA-123075 or a descendant thereof. In someembodiments, the nucleotide sequence is at least 70% homologous, atleast 80% homologous, at least 85% homologous, at least 90% homologous,at least 95% homologous, at least 97% homologous, or at least 99%homologous to the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:6. In some embodiments, the ACCase nucleotidesequence is operably linked to a promoter sequence and forms part of anexpression or inhibition construct, and in some embodiments the ACCasenucleotide sequence is at least 70% homologous, at least 80% homologous,at least 85% homologous, at least 90% homologous, at least 95%homologous, at least 97% homologous, or at least 99% homologous to theACC resistant herbicide gene as found in the AF28-A, AF26-B and/orAF10-D, or SEQ ID NO:4, SEQ ID NO:5 and/or SEQ ID NO:6 comprising anamino acid substitution Ala 2004 Val in the A, B, or D genome.

Classical Breeding of Wheat

Field crops have been classically bred through techniques that takeadvantage of the plants method(s) of pollination. A plant is considered“self-pollinating” if pollen from one flower can be transmitted to thesame or another flower, whereas plants are considered “cross-pollinated”if the pollen has to come from a flower on a different plant in orderfor pollination to occur. Plants that are self-pollinated and selectedover many generations become homozygous at most, if not all, of theirgene loci, thereby producing a uniform population of true breedingprogeny. A cross between two homozygous plants from differingbackgrounds or two different homozygous lines will produce a uniformpopulation of hybrid plants that will more than likely be heterozygousat a number of the gene loci. A cross of two plants that are eachheterozygous at a number of gene loci will produce a generation ofhybrid plants that are genetically different and are not uniform.

Wheat plants are self-pollinating plants, but they can also be bred bycross-pollination. The development of wheat hybrids requires thedevelopment of pollinator parents (fertility restorers) and seed parentinbreds using the cytoplasmic male sterility-fertility restorer system,the crossing of seed parents and pollinator parents, and the evaluationof the crosses. Pedigree breeding programs combine desirable traits; inthe present application the desirable trait being plant resistance toACC herbicides. This trait is put into the breeding pool from one ormore lines, such that new inbred lines are created by crossing, followedby selection of plants with the desired trait, followed by morecrossing, etc. New inbreds are crossed with other inbred lines (e.g.,elite plant lines like those described herein).

Pedigree breeding starts with the crossing of two genotypes, such asAF28-A, AF26-B and/or AF10-D, and an elite wheat line. If the originaltwo parents do not provide all of the desired characteristics, thenother sources can be included in the breeding population. For example,if a hybrid is desired such that both ACC herbicide resistance andresistance to another herbicide group as described herein was desirous,then plants with both these attributes could be crossed using classicalbreeding techniques. In the pedigree method, superior plants are selfedand selected in successive generations. In the succeeding generations,the heterozygous condition gives way to homogeneous lines as a result ofself-pollination and selection. Typically, in the pedigree method, fiveor more generations of selfing and selection are practiced (e.g., S1,S2, S3, S4, S5, etc.).

Backcrossing is used to improve a plant line. Backcrossing transfers aspecific desirable trait from one source to another that lacks thetrait. This is accomplished by, for example, crossing a donor (e.g.,AF28-A) to an elite inbred line (e.g., an elite line). The progeny ofthis cross is then crossed back (i.e. backcrossing) to the elite inbredline, followed by selection in the resultant progeny for the desiredtrait (e.g., resistance to ACC herbicides). Following five or morebackcross generations with selection for the desired trait the progenyare typically heterozygous for the locus (loci) controlling the desiredphenotype, but will be like the elite parent for the other genetictraits. The last backcrossing then is typically selfed in order to givea pure breeding progeny for the gene being transferred.

In current hybrid wheat breeding programs, new parent lines aredeveloped to be either seed-parent lines or pollen-parent linesdepending on whether or not they contain fertility restoring genes; theseed-parent lines do not have fertility restoring genes and aremale-sterile in certain cytoplasms (also known as “A” line plants) andmale-fertile in other cytoplasms (also known as “B” line plants),whereas the pollen-parent lines are not male sterile and do containfertility restoring genes (also known as “R” line plants). Theseed-parent lines are typically created to be cytoplasmically malesterile such that the anthers are minimal to non-existent in theseplants thereby requiring cross-pollination. The seed-parent lines willonly produce seed, and the cytoplasm is transmitted only through theegg. The pollen for cross pollination is furnished through thepollen-parent lines that contain the genes necessary for completefertility restoration in the F1 hybrid, and the cross combines with themale sterile seed parent to produce a high-yielding single cross hybridwith good grain quality.

Typically, this cytoplasmic male sterility-fertility restorer system isperformed for the production of hybrid seed by planting blocks of rowsof male sterile (seed-parent) plants and blocks of rows of fertilityrestorer (pollen-parent) plants, such that the seed-parent plants arewind pollinated with pollen from the pollen-parent plant. This processproduces a vigorous single-cross hybrid that is harvested and planted bythe consumer. Male sterile, seed-parent plants can also be created bygenetically breeding recessive male-sterile nuclear genes into aparticular population, however the cytoplasmic male sterility-fertilityrestorer system is typically the system used for breeding hybrid wheat.Sleper and Poehlman, 2006, Breeding Field Crops, Fifth Ed., BlackwellPublishing provides a good review of current wheat breeding proceduresand is incorporated herein in its entirety.

The present invention is not limited to the wheat lines listed, and oneskilled in the art will recognize that any elite wheat line would beequally amenable to the compositions and methods as described herein.

Plant Transgenics

Compositions of the present invention include the sequences for wheatnucleotide sequences which have been identified as ACCase encodingsequences that are involved in plant response to ACCase herbicides. Inparticular, the present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NOs: 5, 6, 7, 8, and 9. Further provided arepolypeptides having an amino acid sequence encoded by a nucleic acidmolecule described herein, for example those nucleotide sequences setforth in SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

The compositions of the invention can be used in a variety of methodswhereby the protein products can be expressed in crop plants to functionas herbicide resistant proteins. Such expression results in thealteration or modulation of the level, tissue, or timing of expressionto achieve improved resistance to ACCase herbicides. The compositions ofthe invention may be expressed in the same species from which theparticular ACCase originates, or alternatively, can be expressed in anyplant of interest. In this manner, the coding sequence for the ACCasecan be used in combination with a promoter that is introduced into acrop plant. In one embodiment, a high-level expressing constitutivepromoter may be utilized and would result in high levels of expressionof the ACCase. In other embodiments, the coding sequence may be operablylinked to a tissue-specific promoter to direct the expression to a planttissue known to be susceptible to ACCase herbicides such as leaves.Likewise, manipulation of the timing of expression may be utilized. Forexample, by judicious choice of promoter, expression can be enhancedearly in plant growth to prime the plant to be responsive to herbicidetreatment.

In specific embodiments, methods for increasing herbicide tolerance in aplant comprise stably transforming a plant with a DNA constructcomprising a nucleotide sequence of the invention operably linked to apromoter that drives expression in a plant.

Transformed plants, plant cells, plant tissues and seeds thereof areadditionally provided.

The methods of the invention can be used with other methods available inthe art for enhancing other traits in plants. It is recognized that suchsecond nucleotide sequences may be used in either the sense or antisenseorientation depending on the desired outcome.

It is this over-expression of mutant ACCase nucleotide sequences (SEQ IDNO:4, 5, and/or 6) that would be the preferred method of use of themutant nucleotide sequences The various advantages and disadvantages ofusing different promoters to drive such over-expression is well known bythose skilled in the art. However, by way of example, a constitutivepromoter could drive the expression, but a more ideal promoter wouldtarget tissues, such as the leaves.

Sequences of the invention, as discussed in more detail below, encompasscoding sequences, antisense sequences, and fragments and variantsthereof. Expression of the sequences of the invention can be used tomodulate or regulate the expression of corresponding ACCase proteins.The invention encompasses isolated or substantially purified nucleicacid or protein compositions.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.“Fragment” means a portion of the nucleotide sequence or a portion ofthe amino acid sequence and hence protein encoded thereby. Fragments ofa nucleotide sequence may encode protein fragments that retain thebiological activity of the native protein and hence have ACCase-likeactivity and thereby affect herbicide response. Alternatively, fragmentsof a nucleotide sequence that are useful as hybridization probesgenerally do not encode fragment proteins retaining biological activity.Thus, fragments of a nucleotide sequence may range from at least about20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up tothe full-length nucleotide sequence encoding the proteins of theinvention.

A fragment of a ACCase nucleotide sequence that encodes a biologicallyactive portion of a ACCase protein of the invention will encode at least15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up tothe total number of amino acids present in a full-length protein of theinvention.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire ACCase sequencesset forth herein or to fragments thereof are encompassed by the presentinvention. Such sequences include sequences that are orthologs of thedisclosed sequences. “Orthologs” means genes derived from a commonancestral gene and which are found in different species as a result ofspeciation. Genes found in different species are considered orthologswhen their nucleotide sequences and/or their encoded protein sequencesshare substantial identity as defined elsewhere herein. Functions oforthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in, for example, Sambrook. See also Innis et al., eds. (1990)PCR Protocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the ACCase sequences of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook.

Biological activity of the ACCase polypeptides (i.e., influencing theACCase herbicide response) can be assayed by any method known in the artand disclosed herein.

The nucleic acid sequences of the present invention can be expressed ina host cell such as bacteria, yeast, insect, mammalian, or preferablyplant cells. It is expected that those of skill in the art areknowledgeable in the numerous expression systems available forexpression of a nucleic acid encoding a protein of the presentinvention. No attempt to describe in detail the various methods knownfor the expression of proteins in prokaryotes or eukaryotes will bemade.

The sequences of the invention are provided in expression cassettes orDNA constructs for expression in the plant of interest. The cassettewill include 5′ and 3′ regulatory sequences operably linked to a ACCasesequence of the invention. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the ACCase sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a polynucleotide of the invention, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theinvention may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theinvention may be heterologous to the host cell or to each other.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of ACCase in the host cell (i.e., plantor plant cell). Thus, the phenotype of the host cell (i.e., plant orplant cell) is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. Methods are available inthe art for synthesizing plant-preferred genes. See, for example, U.S.Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) NucleicAcids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986) Virology 154:9-20); andhuman immunoglobulin heavy-chain binding protein (BiP), (Macejak et al.(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance transcription canalso be utilized.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones,and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724; and WO Publication Nos. 02/36782. Suchdisclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in the host cell of interest. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 (WO 99/48338 and U.S. Pat. No. 6,072,050); the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); PEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, those disclosed in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; and 5,608,142.

Just as expression of an ACCase polypeptides of the invention may betargeted to specific plant tissues or cell types through the use ofappropriate promoters, it may also be targeted to different locationswithin the cell through the use of targeting information or “targetinglabels”. Unlike the promoter, which acts at the transcriptional level,such targeting information is part of the initial translation product.Depending on the mode of infection of the pathogen or the metabolicfunction of the tissue or cell type, the location of the protein indifferent compartments of the cell may make it more efficacious againsta given pathogen or make it interfere less with the functions of thecell. For example, one may produce a protein preceded by a signalpeptide, which directs the translation product into the endoplasmicreticulum, by including in the construct (i.e. expression cassette)sequences encoding a signal peptide (such sequences may also be calledthe “signal sequence”). The signal sequence used could be, for example,one associated with the gene encoding the polypeptide, or it may betaken from another gene.

There are many signal peptides described in the literature, and they arelargely interchangeable (Raikhel N, Chrispeels M J (2000) Proteinsorting and vesicle traffic. In B Buchanan, W Gruissem, R Jones, eds,Biochemistry and Molecular Biology of Plants. American Society of PlantPhysiologists, Rockville, Md., pp 160-201, herein incorporated byreference). The addition of a signal peptide will result in thetranslation product entering the endoplasmic reticulum (in the processof which the signal peptide itself is removed from the polypeptide), butthe final intracellular location of the protein depends on otherfactors, which may be manipulated to result in localization mostappropriate for the pathogen and cell type. The default pathway, thatis, the pathway taken by the polypeptide if no other targeting labelsare included, results in secretion of the polypeptide across the cellmembrane (Raikhel and Chrispeels, supra) into the apoplast. The apoplastis the region outside the plasma membrane system and includes cellwalls, intercellular spaces, and the xylem vessels that form acontinuous, permeable system through which water and solutes may move.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to affect phenotypic changes in the organism. Thus, anymethod, which provides for effective transformation/transfection may beemployed.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055 and Zhao et al., U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, Sanford et al., U.S. Pat. No.4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also seeWeissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In vitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Frommet al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc.Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapman et al.(Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic. One of skill will recognize that after the recombinantexpression cassette is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of number of standard breeding techniques can beused, depending upon the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plans that would produce theselected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Backcrossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Preferably, plants of the present invention are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.).

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli, however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedP L promoter and N-gene ribosome binding site (Shimatake et al. (1981)Nature 292:128). Examples of selection markers for E. coli include, forexample, genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235 and Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention. Such antimicrobial proteins can be used for anyapplication including coating surfaces to target microbes. In thismanner, target microbes include human pathogens or microorganisms.

Synthesis of heterologous nucleotide sequences in yeast is well known.Sherman, F., et al. (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory is a well-recognized work describing the various methodsavailable to produce a protein in yeast. Two widely utilized yeasts forproduction of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques, radioimmunoassay,or other standard immunoassay techniques.

The nucleotide sequences of the present invention may also be used inthe sense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, preferably greater than about 65% sequenceidentity, more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or composition of thepolypeptides of the present invention in a plant or part thereof.Increasing or decreasing the concentration and/or the composition ofpolypeptides in a plant can affect modulation. For example, increasingthe ratio of polypeptides of the invention to native polypeptides canaffect modulation. The method comprises: introducing a polynucleotide ofthe present invention into a plant cell with a recombinant expressioncassette as described above to obtain a transformed plant cell,culturing the transformed plant cell under appropriate growingconditions, and inducing or repressing expression of a polynucleotide ofthe present invention in the plant for a time sufficient to modulate theconcentration and/or the composition of polypeptides in the plant orplant part.

Increasing the Activity and/or Level of a ACCase Polypeptide

Methods are provided to increase the activity and/or level of the ACCasemutant polypeptides to increase tolerance to ACCase herbicides. Anincrease in the level and/or activity of the ACCase mutant polypeptidecan be achieved by providing to the plant a ACCase polypeptide. Thepolypeptide can be provided by introducing mutant ACCase polypeptideinto the plant, introducing into the plant a nucleotide sequenceencoding a mutant ACCase polypeptide or alternatively by modifying agenomic locus encoding the ACCase polypeptide of the invention.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having enhanced ACCase activity. It is also recognized thatthe methods of the invention may employ a polynucleotide that is notcapable of directing, in the transformed plant, the expression of aprotein or an RNA. Thus, the level and/or activity of a ACCAse mutantpolypeptide may be increased by altering the gene encoding the mutantACCase polypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized plantsthat carry mutations in ACCase genes, where the mutations increaseexpression of the mutant ACCase gene or increase the activity of theencoded polypeptide are provided.

Reducing the Activity and/or Level of an ACCase Polypeptide

Methods are also provided to reduce or eliminate the activity of anACCase polypeptide by transforming a plant cell with an expressioncassette that expresses a polynucleotide that inhibits the expression ofthe ACCase. The polynucleotide may inhibit the expression of the ACCasedirectly, by preventing transcription or translation of the ACC synthasemessenger RNA, or indirectly, by encoding a polypeptide that inhibitsthe transcription or translation of an ACCase gene encoding an ACCasepolypeptide. Methods for inhibiting or eliminating the expression of agene in a plant are well known in the art, and any such method may beused in the present invention to inhibit the expression of the ACCasepolypeptide. Many methods may be used to reduce or eliminate theactivity of an ACC synthase polypeptide. In addition, more than onemethod may be used to reduce the activity of a single ACCasepolypeptide.

1. Polynucleotide-Based Methods:

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of an ACC synthasepolypeptide of the invention. The term “expression” as used hereinrefers to the biosynthesis of a gene product, including thetranscription and/or translation of said gene product. For example, forthe purposes of the present invention, an expression cassette capable ofexpressing a polynucleotide that inhibits the expression of at least oneACC synthase polypeptide is an expression cassette capable of producingan RNA molecule that inhibits the transcription and/or translation of atleast one ACC synthase polypeptide of the invention. The “expression” or“production” of a protein or polypeptide from a DNA molecule refers tothe transcription and translation of the coding sequence to produce theprotein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of an ACCsynthase polypeptide include sense Suppression/Cosuppression, where anexpression cassette is designed to express an RNA molecule correspondingto all or part of a messenger RNA encoding an ACC synthase polypeptidein the “sense” orientation and over expression of the RNA molecule canresult in reduced expression of the native gene; Antisense Suppressionwhere the expression cassette is designed to express an RNA moleculecomplementary to all or part of a messenger RNA encoding the ACCsynthase polypeptide and over expression of the antisense RNA moleculecan result in reduced expression of the native gene; Double-Stranded RNAInterference, where a sense RNA molecule like that described above forcosuppression and an antisense RNA molecule that is fully or partiallycomplementary to the sense RNA molecule are expressed in the same cell,resulting in inhibition of the expression of the correspondingendogenous messenger RNA, Hairpin RNA Interference and Intron-ContainingHairpin RNA Interference, where the expression cassette is designed toexpress an RNA molecule that hybridizes with itself to form a hairpinstructure that comprises a single-stranded loop region and a base-pairedstem, Small Interfering RNA or Micro RNA, where the expression cassetteis designed to express an RNA molecule that is modeled on an endogenousmiRNA gene.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding an ACCase polypeptide, resulting in reducedexpression of the gene, Methods of selecting sites for targeting by zincfinger proteins have been described, for example, in U.S. Patent No.6,453,242, and methods for using zinc finger proteins to inhibit theexpression of genes in plants are described, for example, in U.S. PatentPublication Nos. 2003/0037355; each of which is herein incorporated byreference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one ACCase and reduces the activity ofthe ACC synthase polypeptide. The expression of antibodies in plantcells and the inhibition of molecular pathways by expression and bindingof antibodies to proteins in plant cells are well known in the art. See,for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36,incorporated herein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of an ACCsynthase polypeptide is reduced or eliminated by disrupting the geneencoding the ACC synthase polypeptide. The gene encoding the ACCsynthase polypeptide may be disrupted by any method known in the art.For example, in one embodiment, the gene is disrupted by transposontagging. In another embodiment, the gene is disrupted by mutagenizingplants using random or targeted mutagenesis, and selecting for plantsthat have reduced ACCase activity.

In certain embodiments the nucleic acid sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides of the present invention, (SEQID NOS: 1, 2, 3, 4, 5, or 6), or with other genes implicated inherbicide resistance. The combinations generated can also includemultiple copies of any one of the polynucleotides of interest. Thepolynucleotides of the present invention can also be stacked with anyother gene or combination of genes to produce plants with a variety ofdesired trait combinations including but not limited to traits desirablefor animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,409)); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279;Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) PlantMol. Biol. 12: 123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001));and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001), the disclosures of which are herein incorporated by reference.

The polynucleotides of the present invention can also be stacked withtraits desirable for insect, disease or herbicide resistance (e.g.,Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones et al. (1994) Science 266:789; Martin etal. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene and GAT gene)); and traits desirable forprocessing or process products such as high oil (U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (U.S. Pat. No. 5,602,321); beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847), which facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides providing agronomic traitssuch as male sterility (see U.S. Pat. No. 5,583,210), stalk strength,flowering time, or transformation technology traits such as cell cycleregulation or gene targeting (see, WO 99/61619; WO 00/17364; WO99/25821), the disclosures of which are herein incorporated byreference.

These stacked combinations can be created by any method including, butnot limited to, polynucleotide sequences of interest can be combined atany time and in any order. For example, a transgenic plant comprisingone or more desired traits can be used as the target to introducefurther traits by subsequent transformation. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Genotyping provides a meansof distinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,Springer-Verlag, Berlin (1997). For molecular marker methods, seegenerally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:Genome Mapping in plants (Ed., Andrew H. Paterson) by AcademicPress/R.G. Lands Company, Austin, Tex., pp. 7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentsresulting from nucleotide sequence variability. As is well known tothose of skill in the art, RFLPs are typically detected by extraction ofgenomic DNA and digestion with a restriction enzyme. Generally, theresulting fragments are separated according to size and hybridized witha probe; single copy probes are preferred. Restriction fragments fromhomologous chromosomes are revealed. Differences in fragment size amongalleles represent an RFLP. Thus, the present invention further providesa means to follow segregation of a gene or nucleic acid of the presentinvention as well as chromosomal sequences genetically linked to thesegenes or nucleic acids using such techniques as RFLP analysis. Linkedchromosomal sequences are within 50 centiMorgans (cM), often within 40or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2,or 1 cM of a gene of the present invention.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes hybridize, under selectivehybridization conditions, to a gene encoding a polynucleotide of thepresent invention. In preferred embodiments, the probes are selectedfrom polynucleotides of the present invention. Typically, these probesare cDNA probes or restriction enzyme treated (e.g., PST I) genomicclones. The length of the probes is typically at least 15 bases inlength, more preferably at least 20, 25, 30, 35, 40, or 50 bases inlength. Generally, however, the probes are less than about 1 kilobase inlength. Preferably, the probes are single copy probes that hybridize toa unique locus in a haploid chromosome compliment. Some exemplaryrestriction enzymes employed in RFLP mapping are EcoRI, EcoRV, and SstI.As used herein the term “restriction enzyme” includes reference to acomposition that recognizes and, alone or in conjunction with anothercomposition, cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present invention of the genomicDNA; (c) detecting therefrom a RFLP. Other methods of differentiatingpolymorphic (allelic) variants of polynucleotides of the presentinvention can be had by utilizing molecular marker techniques well knownto those of skill in the art including such techniques as: 1) singlestranded conformation analysis (SSCA); 2) denaturing gradient gelelectrophoresis (DGGE); 3) RNase protection assays; 4) allele-specificoligonucleotides (ASOs); 5) the use of proteins which recognizenucleotide mismatches, such as the E. coli mutS protein; and 6)allele-specific PCR. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); andchemical mismatch cleavage (CMC). Thus, the present invention furtherprovides a method of genotyping comprising the steps of contacting,under stringent hybridization conditions, a sample suspected ofcomprising a polynucleotide of the present invention with a nucleic acidprobe. Generally, the sample is a plant sample, preferably, a samplesuspected of comprising a maize polynucleotide of the present invention(e.g., gene, mRNA). The nucleic acid probe selectively hybridizes, understringent conditions, to a subsequence of a polynucleotide of thepresent invention comprising a polymorphic marker. Selectivehybridization of the nucleic acid probe to the polymorphic markernucleic acid sequence yields a hybridization complex. Detection of thehybridization complex indicates the presence of that polymorphic markerin the sample. In preferred embodiments, the nucleic acid probecomprises a polynucleotide of the present invention.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a nucleotide construct iscomprised of a coding sequence for a protein or an RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ anucleotide construct that is not capable of directing, in a transformedplant, the expression of a protein or an RNA.

In addition, it is recognized that methods of the present invention donot depend on the incorporation of the entire nucleotide construct intothe genome, only that the plant or cell thereof is altered as a resultof the introduction of the nucleotide construct into a cell. In oneembodiment of the invention, the genome may be altered following theintroduction of the nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions, and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions, or substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions, or substitutions comprise at least one nucleotide.

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLES Example 1 An Acetyl Co-Enzyme A Carboxylase Inhibitor TolerantWheat (Triticum Aestivum L.) for Use in a Herbicide Tolerant CroppingSystem

A winter wheat (Triticum aestivum L.) with tolerance to the AcetylCo-Enzyme A Carboxylase (ACCase) inhibitor class of herbicides wasdeveloped via the following method:

Winter wheat seed, variety Hatcher, was subjected to a potent chemicalmutagen (non-transgenic method), ethane methylsulfonate (EMS), at a rateof 0.75% for 2.5 hours. This seed is hereby denoted Ml, each subsequentgeneration of seed will be denoted with a sequentially increasingnumeral following the M. This resulted in a mutation frequency in thewheat genome of about 1 mutation per 96 kb (calculated in the M2generation). This wheat was planted in February and harvested July. Theresulting M2 seed was planted in the field in Sept at a total populationof 2.5 million plants.

In May the following year, the field was divided into two sections; onesection was treated with a lethal dose of quizalofop (-1 million plants)and the other section was treated with a lethal dose of clethodim (-1.5million plants). Quizalofop and clethodim are highly effective ACCaseinhibitors (lipid synthesis inhibitor). The quizalofop portion of thefield was treated a second time in June. 46 quizalofop and 167 clethodimsurvivors' heads were collected from the field July.

Concurrently a small portion of M2 seed was planted in the greenhousefrom Jan-April. Approximately 75,000 and 175,000 plants were screenedwith lethal doses of quizalofop and clethodim respectively. Afterapplication, a small subset of clethodim survivors (7 plants) thatappeared healthier than the rest were screened a second time. This wasthe first documented incidence of improved herbicide tolerance in ourmutant population (FIG. 1), May. Total, 26 quizalofop and 42 clethodimsurvivors were harvested from these sets of plants.

The M3 generation collected from the field has now been screened in thegreenhouse (Aug-Oct) for quizalofop and clethodim with two sequentialrates of a lethal dose of herbicide (FIG. 2). Some accessions exhibiteda high survival rate compared to other mutant plants and theun-mutagenized check. Some preliminary characterization studiesinvestigating the various mutations have begun. FIG. 3 shows some M3ACCase tolerant accessions compared to the un-mutagenized check.

These screenings provide clear evidence that this wheat has acquiredACCase resistance that is inheritable and functional.

Example 2 An Acetyl Co-Enzyme A Carboxylase Inhibitor Tolerant Wheat(Triticum Aestivum L.) for Use in a Herbicide Tolerant Cropping System

A winter wheat (Triticum aestivum L.) with tolerance to the AcetylCo-Enzyme A Carboxylase (ACCase) inhibitor class of herbicides wascharacterized via the following methods:

Plants exhibiting an increased tolerance to quizalofop herbicide werescreened with multiple methods for identifying and characterizing thecause of increase. Plants were screened for visual injury, whole-plantquizalofop tolerance differences, cross-tolerance, and evaluatedgenotypically and enzymatically.

Visual evaluation. 18 quizalofop-tolerant accessions were treated with21.05 g ai ha⁻¹ quizalofop, a discriminating dose based on previousstudies. Plants were evaluated 28 days after treatment (DAT) for visibleinjury to quizalofop on a scale of 0 to 100%, with 0 being no injury and100 being complete desiccation. Nearly all accessions evaluated in thisstudy appeared more tolerant to quizalofop than non-mutant Hatcher wheat(FIG. 5). The accessions had few completely dead plants, with theexception of the one accession not different than the background.

-   Dose response. A dose response study was completed with 11, 23, 46,    92, and 184 g quizalofop ha⁻¹. Seven DAT the tops of plants were cut    off above the newest above-ground growing point. Binomial evaluation    of plant survival was performed 28 DAT. Differences were uncovered    in the whole plant sensitivity to increasing application rates of    quizalofop. LD₅₀'s ranged from 10 g ai ha⁻¹, with the non-treated,    to 76 g ai ha⁻¹ (FIG. 6). Resistant to susceptible ratios for this    experiment ranged from 1.6 to 7.5 based on survival/death of the    plants.-   Cross-resistance. A cross-resistance study was conducted within the    ACCase herbicide mode of action using herbicides normally lethal to    wheat. Clethodim, sethoxydim, and fluazifop were used at rates of    65, 264, and 361 g ai ha⁻¹, plus a treatment of clethodim and 10.5 g    ai ha⁻¹ quizalofop. Seven DAT the tops of plants were cut off above    the newest above-ground growing point. Binomial evaluation of plant    survival was performed 28 DAT. Tolerance of quizalofop mutants to    clethodim and sethoxydim was low (Table 1). The presence of any    cross tolerance presents evidence that stacking resistant ACCase    copies into a single plant could lead to resistance to additional    herbicides. At this stage only a third of the total ACCase in the    plant would have a mutation and contain tolerance to ACCase    inhibitors if the mutation is target-site-based.-   DNA sequencing. DNA was collected from 26 quizalofop-tolerant    phenotypes. Genome-specific primers were developed to amplify    sequences from the A, B, and D ancestral wheat genomes. Sequenced    results were compared to previously cloned non-mutant wheat    sequences to determine any nucleotide substitutions present. When    comparing sequences from non-mutant Hatcher to mutant phenotypes,    three non-synonymous mutations were revealed in the ACCase    carboxyltransferase domain, all at position 2004 in the Alopecurus    myosuroides amino acid numbering system. This mutation on the A    genome was found in eight accessions, on the B genome in nine    accessions, and on the D genome in nine accessions. No accession had    more than one of these SNPs. The mutation was a C to T substitution    resulting in an alanine to valine change (FIG. 7). Each accession    with higher survival than the background contained one of these    SNPs. Based on the chromatograph patterns, the majority these SNPs    are also believed to be homozygous in the plant.-   ACCase enzyme characterization. An in-vitro enzyme assay was    conducted to observe ACCase in conjunction with quizalofop directly    to determine if the presence of ACCase mutations decreases the    ability of herbicides to inhibit ACCase activity. Four quizalofop    concentrations of 0.1, 1, 10, and 100 μM were included in the assay    along with a non-treated treatment. The experiment included four    accessions which included a representative from the three mutations    detected and non-mutagenized wheat. Non-mutagenized winter wheat had    greater sensitivity to quizalofop than the mutant accessions (FIG.    8). The B and D genome SNPs resulted in higher than background    levels of tolerance to quizalofop at the 10 μM, and the A and D    genome SNPs had higher than background tolerance at the 100 μM    concentration, with LSD's (α=0.05) of 14.5 and 21.6 respectively.    Calculated at the 125 level, the resistant to susceptible value for    the A genome was 4.57, the B genome was 3.57 and the D genome was    10.86.

Based on these experiments, the largest factor in plant tolerance toquizalofop is the presence of a SNP at position 2004.

TABLE 1 Quizalofop tolerant mutant survival after application of otherACCase herbicides. Accession 0 is the non-mutant check. Herbicidetreatment Cleth. + Accession Clethodim Sethoxydin Fluazifop quizalofopNos. % % % % 0 0 0 0 0 1 10 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0 00 6 0 0 0 0 7 0 0 0 0 8 25 0 0 0 9 17 0 0 0 10 0 0 0 0 11 0 8 0 0 12 178 0 0 13 0 0 0 0 14 0 0 0 0 15 0 0 0 0 16 0 0 0 0 17 0 0 0 0 LSD = 16

Example 3

In wheat cropping systems, competition with winter annual grass speciessuch as jointed goatgrass (Aegilops cylindrica), downy brome (Bromustectorum), and feral rye (Secale cereale) can be a major problem formanagers. To combat this problem, new technologies and chemistries areneeded in order to give managers multiple options for grass control.Through a forward genetics screen using an induced mutagenesis method,mutant lines of wheat resistant to the ACCase inhibitor quizalofopp-ethyl were previously characterized (Ostlie et al. 2014), and furthercrosses were performed to create single and two-gene breeding lines.Single gene lines carry the previously characterized ACCase mutation onone of the three wheat genomes (A, B, or D). Two gene lines carry theACCase mutation on two of the three genomes (e.g., AB or AD).

During the 2014-2015 growing season, a field crop safety trial wasperformed to assess these lines for relative levels of resistance andperformance under two application timings, applied with and without thesafener cloquintocet-mexyl. The following objectives were the focus ofthe trials:

Determine relative levels of resistance between two-gene lines andsingle-gene lines. Determine which application timing (early or latespring) provided the highest crop safety. Assess whethercloquintocet-mexyl was effective in increasing crop safety in the field,especially for single-gene lines.

Materials & Methods

One quizalofop susceptible line (Hatcher), four two-gene breeding lines(CO14A006 [AB17], CO14A041[AB157], CO14A075 [BD4], CO14A065 [AD91) andthree one-gene parent lines (AF28, AF26, and AF10) were compared. Asplit-split plot design was used in which quizalofop p-ethyl was appliedat 92.5 g ai ha⁻¹ with 1% MSO corresponding to the highest likely labelapplication rate on March 31st at the tillering growth stage, and on May2^(nd) at the jointing growth stage. Cloquintocet safener (10 g ai/ha)was included in the split-split plot design. In the analysis, visualinjury ratings and height were assessed one month before harvest, andgrain yield in grams per plot was also used as a response factor. TheSAS statistical package and proc mixed were used for analysis with atukey-adjusted p-value of 0.05 as the cutoff value. The analysis wassliced by the cloquintocet factor, and comparisons of timing were madewithin lines. FIG. 10 shows the height data as a percent change fromuntreated control. Letters indicate differences within lines at p<0.05.FIG. 11 shows yield data as a percent change from untreated control.Letters indicate differences within lines at p<0.05. FIG. 12 shows thevisual rating data on a scale of 0 (no injury) to 10 (completemortality). Letters indicate differences within lines at p<0.05.

Discussion

1) Single-gene lines showed significant injury compared to the untreatedcontrol for both the March, and May treatment timings, with theexception of AF28, which only showed injury with the May treatmenttiming. All two-gene lines showed much higher levels of crop safety,with only AB157 and AB17 showing crop injury in the May treatmenttiming. The two-gene line AD91 showed the highest level of crop safety,with no crop injury for any treatment combination.

2) The May treatment timing resulted in consistently higher crop injury(measured as reduced height, reduced yield, or increased injury rating)compared to the March treatment timing, indicating that early springapplication, prior to the jointing growth stage, reduced the likelihoodof crop injury.

3) Cloquintocet-mexyl appeared to be very effective in increasing cropsafety. When single-gene lines treated in March and May also receivedcloquintocet-mexyl, they did not show increased injury compared to theuntreated control, with the exception of AF26, which still showed aminor increase in crop injury from the May treatment timing. Alltwo-gene lines showed no crop injury when treated in combination withthe safener cloquintocet-mexyl. These results indicate that single-genelines when treated with quizalofop in conjunction withcloquintocet-mexyl have sufficient crop safety.

Summary

-   -   1) Two-gene lines showed higher levels of crop safety without        the addition of safener. AD91 showed the highest level of crop        safety out of any tested line.    -   2) Early spring applications prior to jointing show the highest        levels of crop safety.    -   3) Cloquintocet-mexyl was effective in increasing crop safety in        a field test, and was highly effective for single-gene lines

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A method of controlling weeds in the vicinity ofa wheat plant having a modified acetyl-CoA carboxylase protein thatconfers resistance to acetyl-CoA carboxylase herbicide, comprising: a.Obtaining one or more acetyl-CoA carboxylase herbicides b. Obtaining oneor more CQC acid safeners, c. applying said one or more acetyl-CoAcarboxylase herbicides and said CQC acid safeners to a field comprisingsaid wheat plant, wherein weed growth is adversely affected by theapplication of said one or more herbicides and growth of said wheathybrid is not adversely affected.
 2. The method of claim 1 wherein saidmodified protein includes Ala2004Val substitution when compared to wildtype and black grass reference sequence (SEQ ID NO:14 or 16).
 3. Themethod of claim 1 wherein said protein is SEQ ID NO:8, 10, or 12 or itsconservatively modified variants.
 4. The method of claim 1, wherein saidwheat plant comprises one or more mutations in the acetyl-CoAcarboxylase gene as found in AF28-A, AF26-B and/or AF10-D, (ATCC Nos.PTA-123074, PTA-123076 and PTA-123075).
 5. The method of claim 1 whereinsaid herbicide is one from the groups of oraryloxyphenoxypropionates,cyclohexanediones, and/or phenylpyrazolin (DENs).
 6. The method of claim1 wherein said herbicide is one or more of alloxydim, butroxydim,cloproxydim, profoxydim, sethoxydim, clefoxydim, clethodim, cycloxydim,tepraloxydim, tralkoxydim, chloraizfop, clodinafop, clofop, cyhalofop,diclofop, fenoxaprop, fenthiaprop, fluazafop-butyl, fluazifop,haloxyfop, isoxapyrifop, metamifop, propaquizafop, quizalofop, trifopand/or pinoxaden.
 7. The method of claim 1 wherein said safener has thefollowing chemical formula:


8. The method of claim 7 wherein said cloquintocet acid is in the formof an agriculturally acceptable salt or ester.
 9. The method of claim 8wherein said salt includes one or more cations selected from sodium,potassium, and the class of organo ammonium cations wherein the organoammonium cations may have from 1 to about 12 carbon atoms.
 10. Themethod of claim 9 wherein said organo ammonium cations includes,isopropyl ammonium, diglycol ammonium (2-(2-aminoethoxyl)ethanolammonium), dimethyl ammonium, diethyl ammonium, triethyl ammonium,monoethanol ammonium, dimethylethanol ammonium, diethanol ammonium,triethanol ammonium, triisopropanol ammonium, tetramethyl ammonium,tetraethylammonium, N,N,N-trimethylethanol ammonium (choline), andN,N-bis-(3-aminopropyl)methyl ammonium (BAPMA).
 11. The method of claim1 wherein said herbicide is administered in an amount of from about 2 μMor less to about 100 μM or more of herbicide concentration.
 12. Themethod of claim 1 wherein said herbicide is administered in an amountform from about 25 grams of active ingredient per hectare.
 13. Themethod of claim 1 wherein said safener is applied in an amount for fromabout 0.1 gram acid equivalent per hectare to about 300 g gram acidequivalent per hectare.
 14. The method of claim 1 wherein said herbicideand safener are to administered as a single composition.
 15. The methodof claim 14 wherein said herbicide safener composition further comprisesan additional active component such as a pesticide, a plant growthregulator, an insecticide, a fungicide or a fungicide.
 16. The method ofclaim 14 wherein said herbicide safener composition further comprises adispersant and/or a surfactant.
 17. The method of claim 14 wherein saidcomposition further comprises an additional safener.
 18. The method ofclaim 17 wherein said additional safener includes one or more ofbenoxacor, benthiocarb, cloquintocet-mexyl, daimuron, dichlormid,dicyclonon, dimepiperate, fenchlorazole-ethyl, fenclorim, flurazole,fluxofenim, furilazole, Harpin proteins, isoxadifen-ethyl,mefenpyr-diethyl, mephenate, MG 191, MON 4660, naphthalic anhydride(NA), oxabetrinil, R29148 and/or N-phenyl-sulfonylbenzoic acid amides.19. The wheat plant of claim 1, wherein said wheat plant comprises thenucleic acid sequence of SEQ ID NO: 4, 5, or
 6. 20. A method ofproducing wheat for commercial production comprising, planting apopulation of two or more wheat plants, said wheat plants having amodified acetyl-CoA carboxylase protein that confers resistance toacetyl-CoA carboxylase herbicide, a. Applying an acetyl-CoA carboxylaseherbicide to said population of plants; wherein weed growth is adverselyaffected by the application of said herbicide and growth of said wheathybrid is not adversely affected; b. applying a CQC acid safener to saidpopulation; c. allowing said plants to pollinate and thereafter d.harvesting resultant seed.
 21. The method of claim 20 wherein saidmodified protein includes Ala2004Val substitution when compared to wildtype and black grass reference sequence (SEQ ID NO:14 or 16).
 22. Themethod of claim 20 wherein said protein is SEQ ID NO:8, 10, or 12 or itsconservatively modified variants.
 23. The method of claim 20, whereinsaid wheat plant comprises one or more of AF28-A, AF26-B and/or AF10-D,(ATCC Nos. PTA-123074, PTA-123076 and PTA-123075) or a descendantthereof.
 24. The method of claim 20 wherein said herbicide is from oneor more groups of oraryloxyphenoxypropionates, cyclohexanediones, and/orphenylpyrazolin (DENs).
 25. The method of claim 20 wherein saidherbicide is one or more of alloxydim, butroxydim, cloproxydim,profoxydim, sethoxydim, clefoxydim, clethodim, cycloxydim, tepraloxydim,tralkoxydim, chloraizfop, clodinafop, clofop, cyhalofop, diclofop,fenoxaprop, fenthiaprop, fluazafop-butyl, fluazifop, haloxyfop,isoxapyrifop, metamifop, propaquizafop, quizalofop, trifop and/orpinoxaden.
 26. The method of claim 20 further comprising the step of: d)repeating steps a and b so that weeds are controlled during the entireseason.
 27. The method of claim 26 wherein said steps a and b arerepeated 2 or more times.
 28. The method of claim 20 wherein saidsafener has the following chemical formula:


29. The method of claim 20 wherein said cloquintocet acid is in the formof an agriculturally acceptable salt or ester.
 30. The method of claim29 wherein said salt includes containing one or more cations selectedfrom sodium, potassium, and the class of organo ammonium cations whereinthe organo ammonium cations may have from 1 to about 12 carbon atoms.31. The method of claim 30 wherein said organo ammonium cationsincludes, isopropyl ammonium, diglycol ammonium(2-(2-aminoethoxyl)ethanol ammonium), dimethyl ammonium, diethylammonium, triethyl ammonium, monoethanol ammonium, dimethylethanolammonium, diethanol ammonium, triethanol ammonium, triisopropanolammonium, tetramethyl ammonium, tetraethylammonium,N,N,N-trimethylethanol ammonium (choline), andN,N-bis-(3-aminopropyl)methyl ammonium (BAPMA).
 32. The method of claim31 further comprising the step of: processing said harvested grain. 33.An herbicide system for producing a crop of wheat comprising: seed of awheat plant having a modified acetyl-CoA carboxylase protein thatconfers resistance to acetyl-CoA carboxylase herbicide, an acetyl-CoAcarboxylase herbicide and a cloquintocet acid safener.
 34. The system ofclaim 33 wherein said system further comprises an additional activecomponent such as a pesticide, a plant growth regulator, an insecticide,a fungicide or a fungicide.
 35. The system of claim 1 wherein saidcomposition further comprises an additional safener.
 36. The system ofclaim 35 wherein said additional safener includes one or more ofbenoxacor, benthiocarb, cloquintocet-mexyl, daimuron, dichlormid,dicyclonon, dimepiperate, fenchlorazole-ethyl, fenclorim, flurazole,fluxofenim, furilazole, Harpin proteins, isoxadifen-ethyl,mefenpyr-diethyl, mephenate, MG 191, MON 4660, naphthalic anhydride(NA), oxabetrinil, 829148 and/or N-phenyl-sulfonylbenzoic acid amides.37. The system of claim 34 wherein said herbicide is from the groups oforaryloxyphenoxypropionates, cyclohexanediones, and/or phenylpyrazolin(DENs).
 38. The system of claim 37 said herbicide is one or more ofalloxydim, butroxydim, cloproxydim, profoxydim, sethoxydim, clefoxydim,clethodim, cycloxydim, tepraloxydim, tralkoxydim, chloraizfop,clodinafop, clofop, cyhalofop, diclofop, fenoxaprop, fenthiaprop,fluazafop-butyl, fluazifop, haloxyfop, isoxapyrifop, metamifop,propaquizafop, quizalofop, trifop and/or pinoxaden.
 39. A method ofcontrolling weeds in the vicinity of a plant having a modifiedacetyl-CoA carboxylase protein that confers resistance to acetyl-CoAcarboxylase herbicide, comprising: a. obtaining one or more acetyl-CoAcarboxylase herbicides b. obtaining one or more CQC acid safeners, c.applying said one or more acetyl-CoA carboxylase herbicides and said CQCacid safeners to a field comprising said plant, wherein weed growth isadversely affected by the application of said one or more herbicides andgrowth of said plant is not adversely affected.
 40. The method of claim39 wherein said modified protein includes Ala2004Val substitution. 41.The method of claim 39 wherein said herbicide is from the groups oforaryloxyphenoxypropionates, cyclohexanediones, and/or phenylpyrazolin(DENs).
 42. The method of claim 39 wherein said herbicide is one or moreof alloxydim, butroxydim, cloproxydim, profoxydim, sethoxydim,clefoxydim, clethodim, cycloxydim, tepraloxydim, tralkoxydim,chloraizfop, clodinafop, clofop, cyhalofop, diclofop, fenoxaprop,fenthiaprop, fluazafop-butyl, fluazifop, haloxyfop, isoxapyrifop,metamifop, propaquizafop, quizalofop, trifop and/or pinoxaden.
 43. Themethod of claim 39 wherein said safener has the following chemicalformula:


44. The method of claim 43 wherein said cloquintocet acid is in the formof a an agriculturally acceptable salt or ester thereof.
 45. The methodof claim 44 wherein said salt includes containing one or more cationsselected from sodium, potassium, and the class of organo ammoniumcations wherein the organo ammonium cations may have from 1 to about 12carbon atoms.
 46. The method of claim 45 wherein said organo ammoniumcations includes, isopropyl ammonium, diglycol ammonium(2-(2-aminoethoxyl)ethanol ammonium), dimethyl ammonium, diethylammonium, triethyl ammonium, monoethanol ammonium, dimethylethanolammonium, diethanol ammonium, triethanol ammonium, triisopropanolammonium, tetramethyl ammonium, tetraethylammonium,N,N,N-trimethylethanol ammonium (choline), andN,N-bis-(3-aminopropyl)methyl ammonium (BAPMA).
 47. The method of claim43 wherein said herbicide is administered in an amount of from about 2μM or less to about 100 μM or more of herbicide concentration.
 48. Themethod of claim 43 wherein said herbicide is administered in an amountform from about 25 grams of active ingredient per hectare.
 49. Themethod of claim 43 wherein said safener is applied in an amount for formabout) can be applied in an amount of from 0.1 gram acid equivalent perhectare to about 300 g gram acid equivalent per hectare